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Oxysterols are interesting molecules due to their dual nature, reflecting beneficial and harmful effects on the body. An issue that still needs to be solved is how slight modification of their structure owing to the location of the additional polar group in the molecules affects their biological activity. With this in mind, we selected three side chain-hydroxylated oxysterols namely: 20(S)-hydroxycholesterol (20(S)-OH), 24(S)-hydroxycholesterol (24(S)-OH), and 27-hydroxycholesterol (27-OH), and examined their behavior in mixtures with the bioactive sphingolipid - sphingomyelin (SM). Our research was based on the Langmuir monolayer technique supplemented with molecular dynamics (MD) and microscopic observation of the films texture (Brewster angle microscopy, BAM, and atomic force microscopy, AFM). Additionally, since 20(S)-hydroxycholesterol has not been studied so far, we thoroughly characterized this oxysterol in one-component monolayers. Our studies showed differences in the interactions of the studied oxysterols and sphingomyelin. Namely, it was found that 20(S)-OH binds to SM, unlike 24(S)-OH and 27-OH, which both weakly interact with SM. This distinct behavior was interpreted within the molecular dynamics as being due to weak intermolecular interactions between 20(S)-OH molecules, which allowed easy incorporation of SM into the 20(S)-OH monolayer. In contrast, the strong oxysterol-oxysterol interactions occurring in monolayers with 24(S)-OH or 27-OH make this process more difficult. This may be important in the process of bone formation/resorption. Other aspects derived from our study are: (i) the tendency of oxysterols to incorporate into lipid rafts (leading to their modification in structure and function), as well as (ii) the formation of multilayer structures, in which oxysterols are arranged in the characteristic forms of "strings of beads", which may facilitate their transport across the membrane., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

T cell receptor (TCR) plays a fundamental role in adaptive immunity, and TCR-T cell therapy holds great promise for treating solid tumors and other diseases. However, there is a noticeable absence of chemical tools tuning TCR activity. In our study, we screened natural sterols for their regulatory effects on T cell function and identified 7-alpha-hydroxycholesterol (7a-HC) as a potent inhibitor of TCR signaling. Mechanistically, 7a-HC promoted membrane binding of CD3ε cytoplasmic domain, a crucial signaling component of the TCR-CD3 complex, through alterations in membrane physicochemical properties. Enhanced CD3ε membrane binding impeded the condensation between CD3ε and the key kinase Lck, thereby inhibiting Lck-mediated TCR phosphorylation. Transient treatments of TCR-T cells with 7a-HC resulted in reduced signaling strength, increased memory cell populations, and superior long-term antitumor functions. This study unveils a chemical regulation of TCR signaling, which can be exploited to enhance the long-term efficacy of TCR-T cell therapy., Competing Interests: Declaration of interests A patent of using 7a-HC treatment in TCR-T therapy has been filed., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A growing body of evidence suggests that oxysterols such as 25-hydroxycholesterol (25HC) are biologically active and involved in many physiological and pathological processes. Our previous study demonstrated that 25HC induces an innate immune response during viral infections by activating the integrin-focal adhesion kinase (FAK) pathway. 25HC produced the proinflammatory response by binding directly to integrins at a novel binding site (site II) and triggering the production of proinflammatory mediators such as tumor necrosis factor-α (TNF) and interleukin-6 (IL-6). 24-(S)-hydroxycholesterol (24HC), a structural isomer of 25HC, plays a critical role in cholesterol homeostasis in the human brain and is implicated in multiple inflammatory conditions, including Alzheimer's disease. However, whether 24HC can induce a proinflammatory response like 25HC in non-neuronal cells has not been studied and remains unknown. The aim of this study was to examine whether 24HC produces such an immune response using in silico and in vitro experiments. Our results indicate that despite being a structural isomer of 25HC, 24HC binds at site II in a distinct binding mode, engages in varied residue interactions, and produces significant conformational changes in the specificity-determining loop (SDL). In addition, our surface plasmon resonance (SPR) study reveals that 24HC could directly bind to integrin αvβ3, with a binding affinity three-fold lower than 25HC. Furthermore, our in vitro studies with macrophages support the involvement of FAK and NFκB signaling pathways in triggering 24HC-mediated production of TNF. Thus, we have identified 24HC as another oxysterol that binds to integrin αvβ3 and promotes a proinflammatory response via the integrin-FAK-NFκB pathway., (© 2023. The Author(s).)

25-hydroxycholesterol (25-HC) is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase during cholesterol metabolism. The aim of this study was to verify whether 25-HC induces oxiapoptophagy in fibroblasts. 25-HC not only decreased the survival of L929 cells, but also increased the number of cells with condensed chromatin and altered morphology. Fluorescence-activated cell sorting results showed that there was a dose-dependent increase in the apoptotic populations of L929 cells upon treatment with 25-HC. 25-HC-induced apoptotic cell death was mediated by the death receptor-dependent extrinsic and mitochondria-dependent intrinsic apoptosis pathway, through the cascade activation of caspases including caspase-8, -9, and -3 in L929 cells. There was an increase in the levels of reactive oxygen species and inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in L929 cells treated with 25-HC. Moreover, 25-HC caused an increase in the expression of beclin-1 and microtubule-associated protein 1A/1B-light chain 3, an autophagy biomarker, in L929 cells. There was a significant decrease in the phosphorylation of protein kinase B (Akt) in L929 cells treated with 25-HC. Taken together, 25-HC induced oxiapoptophagy through the modulation of Akt and p53 cellular signaling pathways in L929 cells.

Oxysterols (OHCs) are hydroxylated cholesterol metabolites that play ubiquitous roles in health and disease. Due to the non-covalent nature of their interactions and their unique partitioning in membranes, the analysis of live-cell, proteome-wide interactions of OHCs remains an unmet challenge. Here, we present a structurally precise chemoproteomics probe for the biologically active molecule 20(S)-hydroxycholesterol (20(S)-OHC) and provide a map of its proteome-wide targets in the membranes of living cells. Our target catalog consolidates diverse OHC ontologies and demonstrates that OHC-interacting proteins cluster with specific processes in immune response and cancer. Competition experiments reveal that 20(S)-OHC is a chemo-, regio- and stereoselective ligand for the protein transmembrane protein 97 (Tmem97/the σ2 receptor), enabling us to reconstruct the 20(S)-OHC-Tmem97 binding site. Our results demonstrate that multiplexed, quantitative analysis of cellular target engagement can expose new dimensions of metabolite activity and identify actionable targets for molecular therapy., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The COVID-19 pandemic caused by SARS-CoV-2 infection poses a serious threat to global public health and the economy. The enzymatic product of cholesterol 25-hydroxylase (CH25H), 25-Hydroxycholesterol (25-HC), was reported to have potent anti-SARS-CoV-2 activity. Here, we found that the combination of 25-HC with EK1 peptide, a pan-coronavirus (CoV) fusion inhibitor, showed a synergistic antiviral activity. We then used the method of 25-HC modification to design and synthesize a series of 25-HC-modified peptides and found that a 25-HC-modified EK1 peptide (EK1P4HC) was highly effective against infections caused by SARS-CoV-2, its variants of concern (VOCs), and other human CoVs, such as HCoV-OC43 and HCoV-229E. EK1P4HC could protect newborn mice from lethal HCoV-OC43 infection, suggesting that conjugation of 25-HC with a peptide-based viral inhibitor was a feasible and universal strategy to improve its antiviral activity.

25-hydroxycholesterol (25-OH), a molecule with unusual behavior at the air/water interface, being anchored to the water surface alternatively with a hydroxyl group at C(3) or C(25), has been investigated in mixtures with main membrane phospholipids (phosphatidylcholines - PCs, and phosphatidylethanolamines - PEs), characteristic of the outer and inner membrane leaflet, respectively. To achieve this goal, the classical Langmuir monolayer approach based on thermodynamic analysis of interactions was conducted in addition to microscopic imaging of films (in situ with BAM and after transfer onto mica with AFM), surface-sensitive spectroscopy (PM-IRRAS), as well as theoretical calculations. Our results show that the strength of interactions is primarily determined by the kind of polar group (strong, attractive interactions leading to surface complexes formation were found to occur with PCs while weak or repulsive ones with PEs). Subsequently, the saturation of phosphatidylcholines apolar chain(s) was found to be crucial for the structure of the formed complexes. Namely, saturated PC (DPPC) does not have preferences regarding the orientation of 25-OH molecule in surface complexes (which results in the two possible 25-OH arrangements), while unsaturated PC (DOPC) enforces one specific orientation of oxysterol (with C(3)-OH group). Our findings suggest that the transport of 25-OH between inner and outer membrane leaflet can proceed without orientation changes, which is thermodynamically advantageous. This explains results found in real systems showing significant differences in the rate of transmembrane transport of 25-OH and the other chain-oxidized oxysterols compared to their ring-oxidized analogues or cholesterol., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The covalent OH bonds of water vibrate and absorb radiation in the near infrared (NIR) region at wavelengths that vary according to the strength of the bonds which, at the same time, are sensitive to the number and/or strength of hydrogen bonds. By means of multivariate analytical tools, such spectral shift was exploited to study the effect of temperature, 25-hydroxycholesterol and progesterone on the H-bonded network of water in DMPA membranes. Temperature was found as the dominating factor altering the NIR spectra of water and then the H-bonds. Increasing temperatures disrupt the H-bonds network, strengthening the OH covalent bonds. The disruption of the H-bonds along the 13-58 °C range was noticeably greater than that caused by lipids or steroids at 500 μM. The H-bonded network of the interfacial water in DMPA membranes was disrupted by the presence of 25-hydroxycholesterol, but no significant disruption was observed in the presence of progesterone. The reduction of the H-bonds entails a reduction in the aggregation of the interfacial water by a reduction in the number of H-bonded molecules. It is proposed that the number of water molecules bonded with two H-bonds diminishes and the number of molecules with no H-bond increases roughly at similar proportions, with a constant population of molecules with one H-bond. The opposed effects of steroids are discussed in the context of their opposed effects on the phase state of membranes, the membrane water content and the steroid molecular structure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The sterol regulatory element-binding protein (SREBP) pathway controls cellular homeostasis of sterols. The key players in this pathway, Scap and Insig-1 and -2, are membrane-embedded sterol sensors. The 25-hydroxycholesterol (25HC)-dependent association of Scap and Insig acts as the master switch for the SREBP pathway. Here, we present cryo-electron microscopy analysis of the human Scap and Insig-2 complex in the presence of 25HC, with the transmembrane (TM) domains determined at an average resolution of 3.7 angstrom. The sterol-sensing domain in Scap and all six TMs in Insig-2 were resolved. A 25HC molecule is sandwiched between the S4 to S6 segments in Scap and TMs 3 and 4 in Insig-2 in the luminal leaflet of the membrane. Unwinding of the middle of the Scap-S4 segment is crucial for 25HC binding and Insig association., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cholesterol is responsible for the plasticity of plasma membranes and is involved in physiological and pathophysiological responses. Cholesterol homeostasis is regulated by oxysterols, such as 25-hydroxycholesterol. The presence of 25-hydroxycholesterol at the membrane level has been shown to interfere with several viruses' entry into their target cells. We used atomic force microscopy to assess the effect of 25-hydroxycholesterol on different properties of supported lipid bilayers with controlled lipid compositions. In particular, we showed that 25-hydroxycholesterol inhibits the lipid-condensing effects of cholesterol, rendering the bilayers less rigid. This study indicates that the inclusion of 25-hydroxycholesterol in plasma membranes or the conversion of part of their cholesterol content into 25-hydroxycholesterol leads to morphological alterations of the sphingomyelin (SM)-enriched domains and promotes lipid packing inhomogeneities. These changes culminate in membrane stiffness variations.

Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts., (Copyright © 2020. Published by Elsevier B.V.)

25-Hydroxycholesterol (25HC), one of the most important oxysterol molecules, can be used by cells to fight bacterial and viral infections but the mechanism that defines its biological effects are unknown. Using molecular dynamics, we have aimed to describe the orientation and location of 25HC in the membrane as well as the interactions it might have with lipids. We have studied two complex model membrane systems, one similar to the late endosome membrane and the other one to the plasma membrane. Our results reinforce that 25HC is inserted in the membrane in a relative stable location similar to but not identical to cholesterol. 25HC fluctuates in the membrane to a much greater degree than cholesterol, but the effect of 25HC on the phospholipid order parameters is not significantly different. One of the most notable facts about 25HC is that, unlike cholesterol, this molecule tends to aggregate, forming dimers, trimers and higher-order aggregates. These aggregates are formed spontaneously through the formation of hydrogen bonds between the two 25HC atoms, the formation of hydrogen bonds being independent of the studied system. Remarkably, no contacts or hydrogen bonds are observed between 25HC and cholesterol molecules, as well as between cholesterol molecules themselves at any time. It would be conceivable that 25HC, by forming high order aggregates without significantly altering the membrane properties, would modify the way proteins interact with the membrane and henceforth form a true innate antiviral molecule., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is an immune-mediated disease characterized by a complex neuropsychiatric syndrome in association with an antibody-mediated decrease of NMDAR. About 85% of patients respond to immunotherapy (and removal of an associated tumour if it applies), but it often takes several months or more than 1 year for patients to recover. There are no complementary treatments, beyond immunotherapy, to accelerate this recovery. Previous studies showed that SGE-301, a synthetic analogue of 24(S)-hydroxycholesterol, which is a potent and selective positive allosteric modulator of NMDAR, reverted the memory deficit caused by phencyclidine (a non-competitive antagonist of NMDAR), and prevented the NMDAR dysfunction caused by patients' NMDAR antibodies in cultured neurons. An advantage of SGE-301 is that it is optimized for systemic delivery such that plasma and brain exposures are sufficient to modulate NMDAR activity. Here, we used SGE-301 to confirm that in cultured neurons it prevented the antibody-mediated reduction of receptors, and then we applied it to a previously reported mouse model of passive cerebroventricular transfer of patient's CSF antibodies. Four groups were established: mice receiving continuous (14-day) infusion of patients' or controls' CSF, treated with daily subcutaneous administration of SGE-301 or vehicle (no drug). The effects on memory were examined with the novel object location test at different time points, and the effects on synaptic levels of NMDAR (assessed with confocal microscopy) and plasticity (long-term potentiation) were examined in the hippocampus on Day 18, which in this model corresponds to the last day of maximal clinical and synaptic alterations. As expected, mice infused with patient's CSF antibodies, but not those infused with controls' CSF, and treated with vehicle developed severe memory deficit without locomotor alteration, accompanied by a decrease of NMDAR clusters and impairment of long-term potentiation. All antibody-mediated pathogenic effects (memory, synaptic NMDAR, long-term potentiation) were prevented in the animals treated with SGE-301, despite this compound not antagonizing antibody binding. Additional investigations on the potential mechanisms related to these SGE-301 effects showed that (i) in cultured neurons SGE-301 prolonged the decay time of NMDAR-dependent spontaneous excitatory postsynaptic currents suggesting a prolonged open time of the channel; and (ii) it significantly decreased, without fully preventing, the internalization of antibody-bound receptors suggesting that additional, yet unclear mechanisms, contribute in keeping unchanged the surface NMDAR density. Overall, these findings suggest that SGE-301, or similar NMDAR modulators, could potentially serve as complementary treatment for anti-NMDAR encephalitis and deserve future investigations., (© The Author(s) (2020). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Oxysterols are molecules derived by the oxidation of cholesterol and can be formed either by auto-oxidation, enzymatically or by both processes. Among the oxysterols formed by auto-oxidation, 7-ketocholesterol and 7β-hydroxycholesterol are the main forms generated. These oxysterols, formed endogenously and brought in large quantities by certain foods, have major cytotoxic properties. They are powerful inducers of oxidative stress, inducing dysfunction of organelles (mitochondria, lysosomes and peroxisomes) that can cause cell death. These molecules are often identified in increased amounts in common pathological states such as cardiovascular diseases, certain eye conditions, neurodegenerative disorders and inflammatory bowel diseases. To oppose the cytotoxic effects of these molecules, it is important to know their biological activities and the signaling pathways they affect. Numerous cell models of the vascular wall, eye, brain, and digestive tract have been used. Currently, to counter the cytotoxic effects of 7-ketocholesterol and 7β-hydroxycholesterol, natural molecules and oils, often associated with the Mediterranean diet, as well as synthetic molecules, have proved effective in vitro. Bioremediation approaches and the use of functionalized nanoparticles are also promising. At the moment, invertebrate and vertebrate models are mainly used to evaluate the metabolism and the toxicity of 7-ketocholesterol and 7β-hydroxycholesterol. The most frequently used models are mice, rats and rabbits. In order to cope with the difficulty of transferring the results obtained in animals to humans, the development of in vitro alternative methods such as organ/body-on-a-chip based on microfluidic technology are hopeful integrative approaches., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effects of 7β-hydroxycholesterol (7βOH) and 25-hydroxycholesterol (25OH) on the phase behavior and the structural properties of the multilamellar vesicles of dipalmitoylphosphatidylcholine (DPPC) bilayers were investigated with comparing to the effects of cholesterol (Chol). Differential scanning calorimetry (DSC) measurements showed these three sterols have slightly but distinctly different effects on the thermal behavior of DPPC bilayers. X-ray diffraction data analysis on DPPC bilayers containing 30 mol% sterols with the aid of molecular volume data estimated by the neutral buoyancy method indicated that the order of apparent occupied surface area per molecule is DPPC/7βOH > DPPC/25OH > DPPC/Chol at both 25 ℃ and 50 ℃, suggesting that the strength of the condensation effect of these sterols follows inversely this order. Based on the findings in this study, we inferred the molecular orientations of Chol, 7βOH, and 25OH in DPPC bilayers., (Copyright © 2020 Elsevier B.V. All rights reserved.)

20-hydroxycholesterol is a signaling oxysterol with immunomodulating functions and, thus, structural analogues with reporter capabilities could be useful for studying and modulating the cellular processes concerned. We have synthesized three new 20-hydroxycholesterol-like pregn-5-en-3β-ol derivatives with fluorescent 7-nitrobenzofurazan (NBD) or Raman-sensitive alkyne labels in their side-chains. In silico computations demonstrated the compounds possess good membrane permeability and can bind within active sites of known 20-hydroxycholesterol targets (e.g. Smoothened and yeast Osh4) and some other sterol-binding proteins (human LXRβ and STARD1; yeast START-kins Lam4S2 and Lam2S2). Having found good predicted membrane permeability and binding to some yeast proteins, we tested the compounds on microorganisms. Fluorescent microscopy indicated the uptake of the steroids by both Saccharomyces cerevisiae and Yarrowia lipolytica, whereas only S. cerevisiae demonstrated conversion of the compounds into 3-O-acetates, likely because 3-O-acetyltransferase Atf2p is present only in its genome. The new compounds provide new options to study the uptake, intracellular distribution and metabolism of sterols in yeast cells as well as might be used as ligands for sterol-binding proteins., Competing Interests: Declaration of Competing Interest Authors claim no conflict of interests., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Steroidal maleic anhydrides were prepared in one step: lithocholic, chenodeoxicholic, deoxicholic, ursocholic, and hyodeoxicholic acid derivatives. Their capability to induce cell death was studied on C6 rat glioma cells, and 7β-hydroxycholesterol was used as positive cytotoxic control. The highest cytotoxicity was observed with lithocholic and chenodeoxicholic acid derivatives (23-(4-methylfuran-2,5-dione)-3α-hydroxy-24-nor-5β-cholane (compound 1a), and 23-(4-methylfuran-2,5-dione)-3α,7α-dihydroxy-24-nor-5β-cholane (compound 1b), respectively), which induce a non-apoptotic mode of cell death associated with mitochondrial membrane potential loss and reactive oxygen species overproduction. No cells with condensed and/or fragmented nuclei, no PARP degradation and no cleaved-caspase-3, which are apoptotic criteria, were observed. Similar effects were found with 7β-hydroxycholesterol. The cell clonogenic survival assay showed that compound 1b was more cytotoxic than compound 1a and 7β-hydroxycholesterol. Compound 1b and 7β-hydroxycholesterol also induce cell cycle modifications. In addition, compounds 1a and 1b, and 7β-hydroxycholesterol favour the formation of large acidic vacuoles revealed by staining with acridine orange and monodansylcadaverine evocating autophagic vacuoles; they also induce an increased ratio of [LC3-II / LC3-I], and modify the expression of mTOR, Beclin-1, Atg12, and Atg5-Atg12 which is are autophagic criteria. The ratio [LC3-II / LC3-I] is also strongly modified by bafilomycin acting on the autophagic flux. Rapamycin, an autophagic inducer, and 3-methyladenine, an autophagic inhibitor, reduce and increase 7β-hydroxycholesterol-induced cell death, respectively, supporting that 7β-hydroxycholesterol induces survival autophagy. Alpha-tocopherol also strongly attenuates 7β-hydroxycholesterol-induced cell death. However, rapamycin, 3-methyladenine, and α-tocopherol have no effect on compounds 1a and 1b-induced cell death. It is concluded that these compounds trigger a non apoptotic mode of cell death, involving the mitochondria and associated with several characteristics of autophagy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Stereoselective interactions are pivotal for molecular recognition between biomolecules and lipid surfaces. The aim of this study was to determine factors differencing molecular interactions between 7-hydroxycholesterol epimers (oxysterols, which excessively appear in pathological processes in human body) and natural membrane phospholipids. Two-component systems of different mutual proportions of 7-hydroxycholesterol (7α-hydroxycholesterol or 7-β-hydroxycholesterol, in short 7α-OH or 7β-OH) and membrane lipids (POPC, DPPC, DPPE, DPPS, SM) were systematically analyzed in artificial membranes modeled as Langmuir monolayers. Classical surface pressure measurements were complemented with direct visualization of films texture both in situ (with Brewster angle microscopy, BAM) and after their transfer onto solid supports (with Atomic Force Microscopy, AFM). Our results clearly show striking differences in surface properties of the studied binary mixtures, emphasizing distinct effects of both 7-hydroxycholesterol epimers on the organization of lipid layers. Systematic study allowed to conclude that the structure of polar head group and interfacial region of the molecule play important role in oxysterol-phospholipid interactions, while the hydrophobic region is significantly less important in this respect., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The fusion between the viral and the target cell membrane is a crucial step in the life cycle of enveloped viruses. The blocking of this process is a well-known therapeutic approach that led to the development of the fusion inhibitor peptide enfuvirtide, clinically used against human immunodeficiency virus (HIV) type 1. Despite this significant advance on viral treatment, the appearance of resistance has limited its clinical use. Such a limitation has led to the development of other fusion inhibitor peptides, such as C34, that present the same structural domain as enfuvirtide (heptad repeat sequence) but have different functional domains (pocket-binding domain in the case of C34 and lipid-binding domain in the case of enfuvirtide). Recently, the antiviral properties of 25-hydroxycholesterol were demonstrated, which boosted the interest in this oxysterol. The combination of two distinct antiviral molecules, C34 and 25-hydroxycholesterol, may help to suppress the emergence of resistant viruses. In this work, we characterized the interaction of the C34-25-hydroxycholesterol conjugate with biomembrane model systems and human blood cells. Lipid vesicles and monolayers with defined lipid compositions were used as biomembrane model systems. The conjugate interacts preferentially with membranes rich in sphingomyelin (a lipid enriched in lipid rafts) and presents a poor partition to membranes composed solely of phosphatidylcholine and cholesterol. We hypothesize that cholesterol causes a repulsive effect that is overcome in the presence of sphingomyelin. Importantly, the peptide shows a preference for human peripheral blood mononuclear cells relative to erythrocytes, which shows its potential to target CD4 + cells. Antiviral activity results against different wild-type and drug-resistant HIV strains further demonstrated the potential of C34-HC as a good candidate for future studies.

Under pathological conditions, cholesterol oxidation products (oxysterols) appear in enhanced concentration in blood and cerebrospinal fluid, which leads to cytotoxic effect, especially in central nervous system. However, the mode of action of oxysterols on the membrane level has not been fully resolved. In this paper we have investigated the interaction between 7α- hydroxycholesterol, 7α-OH (one of the most abundant oxysterol in human body) and two major membrane lipids: sphingomyelin, SM (basic component of lipid rafts and nerve membrane) and 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine, POPC (main phospholipid of mammalian cell membranes). 7α-OH/SM mixtures may mimic pathologically changed lipid raft (ordered phase, L O ) while the SM/POPC system can model its surrounding (liquid-disordered phase, L α ). For our study, the Langmuir monolayer technique (based on registration of the surface pressure/area, π/A isotherms), complemented with surface visualization technique (Brewster angle microscopy, BAM) and theoretical calculations, have been employed. The observed affinity of 7α-OH to SM, which appears to be stronger than in cholesterol/SM system, indicates that cholesterol might be partially replaced in lipid rafts by its oxidized derivative. Its incorporation significantly increases rigidity of the system in relation to normal (cholesterol-containing) raft, which can disturb its proper functioning. On the other hand, the poor effect of this oxysterol on the raft's environment was observed., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The application of 3D NMR experiments and DFT calculations enabled the structure investigation of C-17 epimer of 3-(25-hydroxycholest-5-enyl) acetate is presented. The H-17 and H-20 protons features the same values of 1 H chemical shift, what causes that the structure elucidation require additional resolution enabled by 3D NMR experiments. The NMR experiments and theoretical calculations allowed for: the resonance assignment (3D COSY-HMBC and 3D TOCSY-HSQC techniques), the prediction of spatial structure (3D NOESY-HSQC and 3D ROESY-HSQC experiments), and the precise measurement of heteronuclear coupling constants (3D HSQC-TOCSY spectra with E.COSY-type multiplets)., (Copyright © 2018 Elsevier Inc. All rights reserved.)

OSBP binds, extracts and transfers sterols and phosphatidylinositol-4-phosphate (PI(4)P between liposomes, but the sequence of steps at the membrane surface leading to ligand removal is poorly characterized. In this study, we used dual polarization interferometry (DPI), a label-free surface analytical technique, to characterize the interaction of recombinant, purified OSBP as it flows over immobilized dioleoyl-phosphatidylcholine (DOPC) bilayers containing PI(4)P, cholesterol or 25-hydroxycholesterol. Kinetics of membrane interaction were analyzed for PI(4)P-binding and phosphorylation mutants of OSBP. Wild-type OSBP demonstrated a distinctive association with immobilized DOPC bilayers containing 1-8 mol% PI(4)P that was characterized by initial saturable binding followed by desorption, indicative of PI(4)P extraction. In support of this conclusion, an OSBP mutant with impaired binding and extraction of PI(4)P was stably absorbed to PI(4)P-containing membranes, while a pleckstrin homology domain mutant did not associate with PI(4)P-containing membranes. The inclusion of >2 mol% cholesterol, but not 25-hydroxycholesterol, in membranes, enhanced the absorption of the wild-type OSBP. A phosphomimetic of OSBP with enhanced in vitro sterol binding activity displayed membrane interaction properties similar to wild-type. These real-time flow studies allow us to dissect the association of OSBP with PI(4)P into discrete components; initial recruitment to PI(4)P membranes by the PH domain, detection and extraction of PI(4)P, and desorption due to ligand depletion., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The intestinal tract is constantly exposed to various stimuli. Group 3 innate lymphoid cells (ILC3s) reside in lymphoid organs and in the intestinal tract and are required for immunity to enteric bacterial infection. However, the mechanisms that regulate the ILC3s in vivo remain incompletely defined. Here, we show that GPR183, a chemotactic receptor expressed on murine and human ILC3s, regulates ILC3 migration toward its ligand 7α,25-dihydroxycholesterol (7α,25-OHC) in vitro, and GPR183 deficiency in vivo leads to a disorganized distribution of ILC3s in mesenteric lymph nodes and decreased ILC3 accumulation in the intestine. GPR183 functions intrinsically in ILC3s, and GPR183-deficient mice are more susceptible to enteric bacterial infection. Together, these results reveal a role for the GPR183-7α,25-OHC pathway in regulating the accumulation, distribution, and anti-microbial and tissue-protective functions of ILC3s and define a critical role for this pathway in promoting innate immunity to enteric bacterial infection., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

BackgroundMaintenance of cholesterol homeostasis is crucial for brain development. Brain cholesterol relies on de novo synthesis and is cleared primarily by conversion to 24S-hydroxycholesterol (24S-HC) with brain-specific cholesterol 24-hydroxylase (CYP46A1). We aimed to investigate the impact of hypoxia-ischemia (HI) on brain cholesterol metabolism in the neonatal mice.MethodsPostnatal day 9 C57BL/6 pups were subjected to HI using the Vannucci model. CYP46A1 expression was assessed with western blotting and its cellular localization was determined using immunofluorescence staining. The amount of brain cholesterol, 24S-HC in the cortex and in the serum, was measured with enzyme-linked immunosorbent assay (ELISA).ResultsThere was a transient cholesterol loss at 6 h after HI. CYP46A1 was significantly upregulated at 6 and 24 h following HI with a concomitant increase of 24S-HC in the ipsilateral cortex and in the serum. The serum levels of 24S-HC correlated with those in the brain, as well as with necrotic and apoptotic cell death evaluated by the expression of spectrin breakdown products and cleaved caspase-3 at 6 and 24 h after HI.ConclusionEnhanced cholesterol turnover by activation of CYP46A1 represents disrupted brain cholesterol homeostasis early after neonatal HI. 24S-HC might be a novel blood biomarker for severity of hypoxic-ischemic encephalopathy with potential clinical application.

The aim of this study was to quantitatively and qualitatively assess the content of cholesterol oxidation products in traditional buttermilk after butter production. Cholesterol oxidation products (COP) exhibit a wide spectrum of biological activity, including cytotoxic, carcinogenic, and pro-oxidative properties. Buttermilk has about 2 mg of COP/kg of fat, including 7β-hydroxycholesterol and 5,6α-epoxycholesterol. Buttermilk immediately after production had a relatively high level of 7β-hydroxycholesterol (1.47 mg/kg), which decreased to 0.61 mg/kg after storage for 10 h at 3°C. During storage, the content of 5,6α-epoxycholesterol increased from 0.50 to 1.40 mg/kg. After 10 h of storage, the antioxidant potential of the buttermilk decreased (expressed as radical scavenging ability; change in 1,1-diphenyl-2-picrylhydrazyl = 32.2%). This study showed the presence of COP in fresh and stored buttermilk and the influence of time on changing the direction of cholesterol oxidation., (Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Mammalian cell entry protein (Mce4A) is a member of MCE-family, and is being considered as a potential drug target of Mycobacterium tuberculosis infection because it is required for invasion and latent survival of pathogen by utilizing host's cholesterol. In the present study, we performed molecular docking followed by 100 ns MD simulation studies to understand the mechanism of interaction of Mce4A to the cholesterol derivatives and probucol. The selected ligands, cholesterol, 25-hydroxycholesterol, 5-cholesten-3β-ol-7-one and probucol bind to the predicted active site cavity of Mce4A, and complexes remain stable during entire simulation of 100 ns. In silico studies were further validated by fluorescence-binding studies to calculate actual binding affinity and number of binding site(s). The non-toxicity of all ligands was confirmed on human monocytic cell (THP1) by MTT assay. This work provides a deeper insight into the mechanism of interaction of Mce4A to cholesterol derivatives, which may be further exploited to design potential and specific inhibitors to ameliorate the Mycobacterium pathogenesis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Recently, it was demonstrated that 25-hydroxycholesterol (25HC), an oxidized cholesterol derivative, inhibits human immunodeficiency virus type 1 (HIV) entry into its target cells. However, the mechanisms involved in this action have not yet been established. The aim of this work was to study the effects of 25HC in biomembrane model systems and at the level of HIV fusion peptide (HIV-FP). Integration of different biophysical approaches was made in the context of HIV fusion process, to clarify the changes at membrane level due to the presence of 25HC that result in the suppressing of viral infection. Lipid vesicles mimicking mammalian and HIV membranes were used on spectroscopy assays and lipid monolayers in surface pressure studies. Peptide-induced lipid mixing assays were performed by Förster resonance energy transfer to calculate fusion efficiency. Liposome fusion is reduced by 50% in the presence of 25HC, comparatively to cholesterol. HIV-FP conformation was assessed by infrared assays and it relies on sterol nature. Anisotropy, surface pressure and dipole potential assays indicate that the conversion of cholesterol in 25HC leads to a loss of the cholesterol modulating effect on the membrane. With different biophysical techniques, we show that 25HC affects the membrane fusion process through the modification of lipid membrane properties, and by direct alterations on HIV-FP structure. The present data support a broad antiviral activity for 25HC., (Copyright © 2018. Published by Elsevier B.V.)

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Oxysterols, oxygenated by-products of cholesterol biosynthesis, play roles in various physiological and pathological systems. However, the effects of oxysterols on periodontal regeneration are unknown. This study investigated the effects of the specific oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on the regeneration of periodontal tissues using in-vitro periodontal ligament stem cells (PDLSCs) and in-vivo models of alveolar bone defect., Methods: To evaluate the effects of the combined oxysterols on PDLSC biology, we studied the SS-induced osteogenic differentiation of PDLSCs by assessing alkaline phosphatase activity, intracellular calcium levels [Ca 2+ ] i , matrix mineralization, and osteogenic marker mRNA expression and protein levels. To verify the effect of oxysterols on alveolar bone regeneration, we employed tooth extraction bone defect models., Results: Oxysterols increased the osteogenic activity of PDLSCs compared with the control group. The expression of liver X receptor (LXR) α and β, the nuclear receptors for oxysterols, and their target gene, ATP-binding cassette transporter A1 (ABCA1), increased significantly during osteogenesis. Oxysterols also increased protein levels of the hedgehog (Hh) receptor Smo and the transcription factor Gli1. We further confirmed the reciprocal reaction between the LXRs and Hh signaling. Transfection of both LXRα and LXRβ siRNAs decreased Smo and Gli1 protein levels. In contrast, the inhibition of Hh signaling attenuated the LXRα and LXRβ protein levels. Subsequently, SS-induced osteogenic activity of PDLSCs was suppressed by the inhibition of LXRs or Hh signaling. The application of SS also enhanced bone formation in the defect sites of in-vivo models, showing equivalent efficacy to recombinant human bone morphogenetic protein-2., Conclusions: These findings suggest that a specific combination of oxysterols promoted periodontal regeneration by regulating PDLSC activity and alveolar bone regeneration.

Dravet syndrome is an infant-onset epileptic encephalopathy with multiple seizure types that are often refractory to conventional therapies. Treatment with standard benzodiazepines like clobazam, in combination with valproate and stiripentol, provides only modest seizure control. While benzodiazepines are a first-line therapy for Dravet syndrome, they are limited by their ability to only modulate synaptic receptors. Unlike benzodiazepines, neuroactive steroids potentiate a wider-range of GABA A receptors. The synthetic neuroactive steroid SGE-516 is a potent positive allosteric modulator of both synaptic and extrasynaptic GABA A receptors. Prior work demonstrated anticonvulsant activity of SGE-516 in acute seizure assays in rodents. In this study, we evaluated activity of SGE-516 on epilepsy phenotypes in the Scn1a +/- mouse model that recapitulates many features of Dravet syndrome, including spontaneous seizures, premature death and seizures triggered by hyperthermia. To evaluate SGE-516 in Scn1a +/- mice, we determined the effect of treatment on hyperthermia-induced seizures, spontaneous seizure frequency and survival. SGE-516 treatment protected against hyperthermia-induced seizures, reduced spontaneous seizure frequency and prolonged survival in the Scn1a +/- mice. This provides the first evidence of SGE-516 activity in a mouse model of Dravet syndrome, and supports further investigation of neuroactive steroids as potential anticonvulsant compounds for refractory epilepsies.

Measles virus (MV) infection is re-emerging, despite the availability of an effective vaccine. The mechanism of MV entry into a target cell relies on coordinated action between the MV hemagglutinin (H) receptor binding protein and the fusion envelope glycoprotein (F) which mediates fusion between the viral and cell membranes. Peptides derived from the C -terminal heptad repeat (HRC) of F can interfere with this process, blocking MV infection. As previously described, biophysical properties of HRC-derived peptides modulate their antiviral potency. In this work, we characterized a MV peptide fusion inhibitor conjugated to 25-hydroxycholesterol (25HC), a cholesterol derivative with intrinsic antiviral activity, and evaluated its interaction with membrane model systems and human blood cells. The peptide (MV.

Capillary electrophoresis (CE) can provide high separation efficiency with very simple instrumentation, but has yet to be explored regarding oxysterols/cholesterol. Cholesterol and 25-hydroxycholesterol (both are 4-ene-3-ketosteroids) were quantitatively transformed into hydrazones using Girard P reagent after enzymatic oxidation by cholesterol oxidase. Separation was achieved using non-aqueous capillary electrophoresis with UV detection at 280nm; the "charge-tagging" Girard P reagent ensured both charge and chromophore (which are requirements for CE-UV). Excess reagent was also separated from the two analytes, eliminating the need for removal prior to the analysis. The compounds were separated in less than 5min with excellent separation efficiency, using separation electrolytes fully compatible with mass spectrometry. The CE-UV method was used to optimize steps for charge-tagging, revealing that the procedure is affected by the analyte/reagent ratio and reaction time, but also the analyte structure., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present in vitro study, for the first time, demonstrates the production of 25-hydroxycholestrol (25-HC) by testicular macrophages of a non-mammalian vertebrate. The ether extracts of testicular macrophage-conditioned medium (TMCM) were fractionated on a C18 reversed phase high-performance liquid chromatography (HPLC) column using methanol as the mobile phase. The mass spectrometry (MS) fragmentation pattern of HPLC-purified 25-HC was found to be identical to that of authentic 25-HC. Further, a significant seasonal variation in 25-HC concentration was observed with maximal level in regressed and minimal during breeding phase. To understand the hormonal control of 25-HC production, testicular macrophages from regressed phase testes were incubated with 0.5μg/ml of ovine follicle stimulating hormone (FSH) and 0.1, 1 and 10μg/ml of testosterone (T). FSH considerably enhanced 25-HC production by testicular macrophages. In contrast, T markedly inhibited 25-HC production in a dose-dependent manner. In addition, T significantly inhibited FSH-induced 25-HC production, though pre-treatment with T was more effective as compared to post-treatment with T to FSH. Our findings on production, seasonal variation and hormonal control of 25-HC suggest the functional significance of 25-HC in the testis of reptiles., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Exosomes from cancer cells are rich sources of biomarkers and may contain elevated levels of lipids of diagnostic value. 27-Hydroxycholesterol (27-OHC) is associated with proliferation and metastasis in estrogen receptor positive (ER+) breast cancer. In this study, we investigated the levels of 27-OHC, and other sidechain-hydroxylated oxysterols in exosomes. To study both cytoplasmic and exosomal oxysterol samples of limited size, we have developed a capillary liquid chromatography-mass spectrometry platform that outperforms our previously published systems regarding chromatographic resolution, analysis time and sensitivity. In the analyzed samples, the quantified level of cytoplasmic 27-OHC using this platform fitted with mRNA levels of 27-OHC's corresponding enzyme, CYP27A1. We find clearly increased levels of 27-OHC in exosomes (i.e., enrichment) from an ER+ breast cancer cell line (MCF-7) compared to exosomes derived from an estrogen receptor (ER-) breast cancer cell line (MDA-MB-231) and other control exosomes (non-cancerous cell line (HEK293) and human pooled serum). The exosomal oxysterol profile did not reflect cytoplasmic oxysterol profiles in the cells of origin; cytoplasmic 27-OHC was low in ER+ MCF-7 cells while high in MDA-MB-231 cells. Other control cancer cells showed varied cytoplasmic oxysterol levels. Hence, exosome profiling in cancer cells might provide complementary information with the possibility of diagnostic value., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Skin photoaging due to UV irradiation is a degenerative process that appears more and more as a growing concern. Lipids, including oxysterols, are involved in degenerative processes; as skin cells contain various lipids, the aim of our study was to evaluate first, changes in keratinocyte lipid levels induced by UV exposure and second, cellular effects of oxysterols in cell morphology and several hallmarks of keratinocyte differentiation. Our mass spectrometry results demonstrated that UV irradiation induces changes in lipid profile of cultured keratinocytes; in particular, ceramides and oxysterols, specifically 25-hydroxycholesterol (25-OH), were increased. Using holography and confocal microscopy analyses, we highlighted cell thickening and cytoskeletal disruption after incubation of keratinocytes with 25-OH. These alterations were associated with keratinocyte differentiation patterns: autophagy stimulation and intracellular calcium increase as measured by cytofluorometry, and increased involucrin level detected by immunocytochemistry. To conclude, oxysterol deregulation could be considered as a common marker of degenerative disorders. During photoaging, 25-OH seems to play a key role inducing morphological changes and keratinocyte differentiation., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Liver X receptors (LXRs) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism. The ability of LXRs to integrate metabolic and inflammation signalling makes them attractive targets for intervention in human metabolic diseases. Several oxidized metabolites of cholesterol (oxysterols) are endogenous LXR ligands, that modulate their transcriptional responses. While 25R-cholestenoic acid is an agonist of the LXRs, the synthetic analogue 27-norcholestenoic acid that lacks the 25-methyl is an inverse agonist. This change in the activity profile is triggered by a disruption of a key interaction between residues His435 and Trp457 that destabilizes the H11-H12 region of the receptor and favors the binding of corepressors. The introduction of fluorine atoms on the oxysterol side chain can favor both hydrophobic interactions as well as hydrogen bonds with the fluorine atoms and may thus induce changes in the receptor that may lead to changes in the activity profile. To evaluate these effects we have synthesized two fluorinated 27-nor-steroids, analogues of 27-norcholestenoic acid, the 25,25-difluoroacid and the corresponding 26-alcohol. The key step was a Reformatsky reaction on the C-24 cholenaldehyde, with ethyl bromodifluoroacetate under high intensity ultrasound (HIU) irradiation, followed by a Barton-McCombie type deoxygenation. Activity was evaluated in a luciferase reporter assay in the human HEK293T cells co-transfected with full length human LXRβ expression vector. The 25,25-difluoro-27-norcholestenoic acid was an inverse agonist and antagonist similar to its non-fluorinated analogue while its reduced derivative 25,25-difluoro-27-norcholest-5-ene-3β,26-diol was an agonist. Molecular dynamics simulation of the ligand-receptor complexes showed that the difluoroacid disrupted the His435-Trp457 interaction although the resulting conformational changes were different from those induced by the non-fluorinated analogue. In the case of the difluoroalcohol, the fluorine atoms actively participated in the interaction with several residues in the ligand binding pocket leading to a stabilization of the active receptor conformation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this work we studied the effect of polyunsaturated fatty acids (PUFAs) methyl esters on cholesterol photo-induced oxidation. The oxidative routes were modeled with a chemical reaction network (CRN), which represents the first application of CRN to the oxidative degradation of a food-related lipid matrix. Docosahexaenoic acid (DHA, T-I), eicosapentaenoic acid (EPA, T-II) and a mixture of both (T-III) were added to cholesterol using hematoporphyrin as sensitizer, and were exposed to a fluorescent lamp for 48h. High amounts of Type I cholesterol oxidation products (COPs) were recovered (epimers 7α- and 7β-OH, 7-keto and 25-OH), as well as 5β,6β-epoxy. Fitting the experimental data with the CRN allowed characterizing the associated kinetics. DHA and EPA exerted different effects on the oxidative process. DHA showed a protective effect to 7-hydroxy derivatives, whereas EPA enhanced side-chain oxidation and 7β-OH kinetic rates. The mixture of PUFAs increased the kinetic rates several fold, particularly for 25-OH. With respect to the control, the formation of β-epoxy was reduced, suggesting potential inhibition in the presence of PUFAs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The availability of reliable assays for measuring 4β-hydroxycholesterol (4β-HC), a CYP3A metabolite of cholesterol, is an important step in qualifying this endogenous moiety as a biomarker of CYP3A activity. Liquid and gas chromatographic methods with mass spectrometric detection have been developed with varying sensitivities, with or without derivatization. Care must be taken to chromatographically resolve 4β-HC from the multiple isobaric cholesterol oxidation products present in plasma, including 4α-hydroxycholesterol (4α-HC). Plasma concentrations of 4β-HC are low in humans (10-60 ng/ml), lower than many other cholesterol metabolites and far less than cholesterol itself. Stability of 4β-HC has been established for at least 12 months at -20°C in plasma samples obtained with a typical clinical workflow. Oxidation of plasma cholesterol during storage produces both 4β-HC and 4α-HC, and 4α-HC may be used as assessment of sample quality. As 4β-HC concentrations over time in untreated individuals have low intra-individual variability, assay precision and reproducibility are the key assay attributes in assessing CYP3A4 induction, and potentially inhibition. Assessment of CYP3A4/5 activity with 4β-HC relies on the differences between pre- and post-dose concentrations, in which each subject acts as their own control. To reduce analytical variability, samples from a single subject should be analyzed together to facilitate interpretation of study results. As an endogenous biomarker, 4β-HC offers the opportunity for less invasive assessment of CYP3A induction potential of new drugs during clinical development.

The DAF-12 receptor in nematodes and the Liver X Receptor (LXR) in mammals are structurally related transcription factors that play key roles in determining the life span of the organism. Both types of receptors are activated by oxysterols, cholesterol metabolites with oxidized side chains. Restricting the movement of the oxysterol side chain to certain orientations may have profound effects in the activity profile, however this has not been explored so far. In a first attempt to obtain analogues of natural ligands of DAF-12 and LXR with restricted side chain mobility we introduced a 16,22-oxygen bridge in 26-hydroxycholesterol, a cholestenoic acid and a dafachronic acid (5-7). Diosgenin was used as starting material, the key step to obtain the 16,22 epoxy functionality was the one pot formation and reduction of a cyclic hemiketal via the oxocarbenium ion using sodium cyanoborohydride. All new compounds were characterized by NMR and mass spectrometry and assayed as ceDAF-12 or LXR ligands in transactivation cell-based assays. The dafachronic acid analogue 7 behaved as a ceDAF-12 agonist., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We synthesized several candidates of 24(S)-hydroxycholesterol (24S-OHC) esters, which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1). We studied the regioselectivity of the acylation of the secondary alcohol at the 3- or 24-position of 24S-OHC. The appropriate saturated and unsaturated long-chain fatty acids were esterified with the protected 24S-OHC and then de-protected to afford the desired esters at a satisfactory yield. We then confirmed by HPLC monitoring that the retention times of four esters of 24S-OHC, namely 3-oleate, 3-linoleate, 3-arachidonoate and 3-docosahexaenoate, were consistent with those of 24S-OHC esters observed in 24S-OHC-treated SH-SY5Y cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Autotaxin (ATX) generates the lipid mediator lysophosphatidic acid (LPA). ATX-LPA signalling is involved in multiple biological and pathophysiological processes, including vasculogenesis, fibrosis, cholestatic pruritus and tumour progression. ATX has a tripartite active site, combining a hydrophilic groove, a hydrophobic lipid-binding pocket and a tunnel of unclear function. We present crystal structures of rat ATX bound to 7α-hydroxycholesterol and the bile salt tauroursodeoxycholate (TUDCA), showing how the tunnel selectively binds steroids. A structure of ATX simultaneously harbouring TUDCA in the tunnel and LPA in the pocket, together with kinetic analysis, reveals that bile salts act as partial non-competitive inhibitors of ATX, thereby attenuating LPA receptor activation. This unexpected interplay between ATX-LPA signalling and select steroids, notably natural bile salts, provides a molecular basis for the emerging association of ATX with disorders associated with increased circulating levels of bile salts. Furthermore, our findings suggest potential clinical implications in the use of steroid drugs.

Objective: Oxidised low density lipoprotein (oxLDL) contributes to atherosclerosis, whereas high density lipoprotein (HDL) is known to be atheroprotective due, at least in part, to its ability to remove oxidised lipids from oxLDL. The molecular details of the lipid transfer process are not fully understood. We aimed to identify major oxidised lipid species of oxLDL and investigate their transfer upon co-incubation with HDL with varying levels of oxidation., Approach and Results: A total of 14 major species of oxidised phosphatidylcholine and oxidised cholesteryl ester from oxLDL were identified using an untargeted mass spectrometry approach. HDL obtained from pooled plasma of normolipidemic subjects (N=5) was oxidised under mild and heavy oxidative conditions. Non-oxidised (native) HDL and oxidised HDL were co-incubated with oxLDL, re-isolated and lipidomic analysis was performed. Lipoprotein surface lipids, oxidised phosphatidylcholines and oxidised cholesterols (7-ketocholesterol and 7β-hydroxycholesterol), but not internal oxidised cholesteryl esters, were effectively transferred to native HDL. Saturated and monounsaturated lyso-phosphatidylcholines were also transferred from the oxLDL to native HDL. These processes were attenuated when HDL was oxidised under mild and heavy oxidative conditions. The impaired capacities were accompanied by an increase in a ratio of sphingomyelin to phosphatidylcholine and a reduction in phosphatidylserine content in oxidised HDL, both of which are potentially important regulators of the oxidised lipid transfer capacity of HDL., Conclusions: Our study has revealed the differential transfer efficiency of surface and internal oxidised lipids from oxLDL and their acceptance onto HDL. These capacities were modulated when HDL was itself oxidised., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cytochrome P450scc (CYP 11A1) catalyzes the conversion of cholesterol (Ch) to pregnenolone, the precursor to steroid hormones. This process proceeds via three sequential monooxygenation reactions: two hydroxylations of Ch first form 22(R)-hydroxycholesterol (HC) and then 20α,22(R)-dihydroxycholesterol (DHC); a lyase reaction then cleaves the C20-C22 bond to form pregnenolone. Recent cryoreduction/annealing studies that employed electron paramagnetic resonance (EPR)/electron nuclear double resonance (ENDOR) spectroscopy [Davydov, R., et al. (2012) J. Am. Chem. Soc. 134, 17149] showed that compound I (Cpd I) is the active intermediate in the first step, hydroxylation of Ch. Herein, we have employed EPR and ENDOR spectroscopy to characterize the intermediates in the second and third steps of the enzymatic process, as conducted by 77 K radiolytic one-electron cryoreduction and subsequent annealing of the ternary oxy-cytochrome P450scc complexes with HC and DHC. This procedure is validated by showing that the cryoreduced ternary complexes of oxy-cytochrome P450scc with HC and DHC are catalytically competent and during annealing generate DHC and pregnenolone, respectively. Cryoreduction of the oxy-P450scc-HC ternary complex trapped at 77K produces the superoxo-ferrous P450scc intermediate along with a minor fraction of ferric hydroperoxo intermediates. The superoxo-ferrous intermediate converts into a ferric-hydroperoxo species after annealing at 145 K. During subsequent annealing at 170-180 K, the ferric-hydroperoxo intermediate converts to the primary product complex with the large solvent kinetic isotope effect that indicates Cpd I is being formed, and (1)H ENDOR measurements of the primary product formed in D2O demonstrate that Cpd I is the active species. They show that the primary product contains Fe(III) coordinated to the 20-O(1)H of DHC with the (1)H derived from substrate, the signature of the Cpd I reaction. Hydroperoxo ferric intermediates are the primary species formed during cryoreduction of the oxy-P450scc-DHC ternary complex, and they decay at 185 K with a strong solvent kinetic isotope effect to form low-spin ferric P450scc. Together, these observations indicated that Cpd I also is the active intermediate in the C20,22 lyase final step. In combination with our previous results, this study thus indicates that Cpd I is the active species in each of the three sequential monooxygenation reactions by which P450scc catalytically converts Ch to pregnenolone.

Cataracts reduce vision in 50% of individuals over 70 years of age and are a common form of blindness worldwide. Cataracts are caused when damage to the major lens crystallin proteins causes their misfolding and aggregation into insoluble amyloids. Using a thermal stability assay, we identified a class of molecules that bind α-crystallins (cryAA and cryAB) and reversed their aggregation in vitro. The most promising compound improved lens transparency in the R49C cryAA and R120G cryAB mouse models of hereditary cataract. It also partially restored protein solubility in the lenses of aged mice in vivo and in human lenses ex vivo. These findings suggest an approach to treating cataracts by stabilizing α-crystallins., (Copyright © 2015, American Association for the Advancement of Science.)

We attempted to determine the effects of a milieu rich in cholesterol molecules on expression of chemokine CXCL8. A high-cholesterol diet led to an increased transcription of the IL-8 gene in the arteries and elevated levels of CXCL8 in sera of ApoE(-/-) mice, compared with those of wild-type C57BL/6 mice. Treatment of THP-1 monocyte/macrophage cells with 27-hydroxycholesterol (27OHChol) resulted in transcription of the IL-8 gene and increased secretion of its corresponding gene product whereas cholesterol did not induce expression of CXCL8 in THP-1 cells. 27OHChol-induced transcription of the IL-8 gene was blocked by cycloheximide, but not by polymyxin B. Treatment of THP-1 cells with 27OHChol caused translocation of p65 NF-κB subunit into the nucleus and up-regulation of CD88. Inhibition of NF-κB and CD88 using SN50 and W-54011, respectively, resulted in reduced transcription of the IL-8 gene and attenuated secretion of CXCL8 induced by 27OHChol. We propose that oxidatively modified cholesterol like 27OHChol, rather than cholesterol, is responsible for sustained expression of CXCL8 in monocytes/macrophages in atherosclerotic arteries., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Insulin-induced gene 1 (Insig-1) and Insig-2 are endoplasmic reticulum membrane-embedded sterol sensors that regulate the cellular accumulation of sterols. Despite their physiological importance, the structural information on Insigs remains limited. Here we report the high-resolution structures of MvINS, an Insig homolog from Mycobacterium vanbaalenii. MvINS exists as a homotrimer. Each protomer comprises six transmembrane segments (TMs), with TM3 and TM4 contributing to homotrimerization. The six TMs enclose a V-shaped cavity that can accommodate a diacylglycerol molecule. A homology-based structural model of human Insig-2, together with biochemical characterizations, suggest that the central cavity of Insig-2 accommodates 25-hydroxycholesterol, whereas TM3 and TM4 engage in Scap binding. These analyses provide an important framework for further functional and mechanistic understanding of Insig proteins and the sterol regulatory element-binding protein pathway., (Copyright © 2015, American Association for the Advancement of Science.)

The behavior of oxysterols in phospholipid membranes and their effects on membrane properties were investigated by means of dynamic light scattering, fluorescence spectroscopy, NMR, and extensive atomistic simulations. Two families of oxysterols were scrutinized-tail-oxidized sterols, which are mostly produced by enzymatic processes, and ring-oxidized sterols, formed mostly via reactions with free radicals. The former family of sterols was found to behave similar to cholesterol in terms of molecular orientation, roughly parallel to the bilayer normal, leading to increasing membrane stiffness and suppression of its membrane permeability. In contrast, ring-oxidized sterols behave quantitatively differently from cholesterol. They acquire tilted orientations and therefore disrupt the bilayer structure with potential implications for signaling and other biochemical processes in the membranes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

We report a straightforward sample preparation procedure and a direct liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the analysis of 7alpha,25-dihydroxycholesterol (7α25-OHC) and 7alpha,27-dihydroxycholesterol (7α27-OHC). By applying a slow protein precipitation approach using cold ethanol, we were able to detect and quantify 7α25-OHC and 7α27-OHC in a fast and reliable manner. The average concentrations from 20 healthy individuals were determined to be 0.21±0.05nM for 7α25-OHC and 3.4±0.1nM for 7α27-OHC. In addition, we are the first to report the average degrees of esterification (n=8) to be 73.8% and 82% for 7α25-OHC and 7α27-OHC, respectively. Using the established method, we achieved the sensitivity sufficient for detecting low abundant dihydroxylated oxysterols in healthy individuals. This result should enable extension of these studies towards a comprehensive analysis of oxysterol levels under disease conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background and Purpose: Memantine and ketamine are clinically used, open-channel blockers of NMDA receptors exhibiting remarkable pharmacodynamic similarities despite strikingly different clinical profiles. Although NMDA channel gating constitutes an important difference between memantine and ketamine, it is unclear how positive allosteric modulators (PAMs) might affect the pharmacodynamics of these NMDA blockers., Experimental Approach: We used two different PAMs: SGE-201, an analogue of an endogenous oxysterol, 24S-hydroxycholesterol, along with pregnenolone sulphate (PS), to test on memantine and ketamine responses in single cells (oocytes and cultured neurons) and networks (hippocampal slices), using standard electrophysiological techniques., Key Results: SGE-201 and PS had no effect on steady-state block or voltage dependence of a channel blocker. However, both PAMs increased the actions of memantine and ketamine on phasic excitatory post-synaptic currents, but neither revealed underlying pharmacodynamic differences. SGE-201 accelerated the re-equilibration of blockers during voltage jumps. SGE-201 also unmasked differences among the blockers in neuronal networks - measured either by suppression of activity in multi-electrode arrays or by neuroprotection against a mild excitotoxic insult. Either potentiating NMDA receptors while maintaining the basal activity level or increasing activity/depolarization without potentiating NMDA receptor function is sufficient to expose pharmacodynamic blocker differences in suppressing network function and in neuroprotection., Conclusions and Implications: Positive modulation revealed no pharmacodynamic differences between NMDA receptor blockers at a constant voltage, but did expose differences during spontaneous network activity. Endogenous modulator tone of NMDA receptors in different brain regions may underlie differences in the effects of NMDA receptor blockers on behaviour., (© 2014 The British Pharmacological Society.)

Objective: The cluster of differentiation-1d (CD1d) recognizes and presents the lipid antigens to NK-T lymphocytes. Atherosclerotic lesions contain atherogenic lipids, mainly cholesterol and its oxides. Peroxisome proliferator-activated receptor-γ (PPARγ) is also known to exist in atherosclerotic lesions, participating in regulation of lipid metabolism. The current study tested whether CD1d acts as a surface receptor that mediates induction and activation of PPARγ by oxysterols commonly found in atherosclerotic lesions., Methods and Results: CD1d overexpression in HEK 293 cells transfected with CD1d cDNA was confirmed by fluorescence, flow cytometry, Western blotting and mRNA expression. Tritiated ((3)H) 7-ketocholesterol (7K) was used for lipid binding assays. Radioactive assessment demonstrated an increased 7K-binding activity HEK 293 cells with CD1d overexpression. The 7K binding could be blocked by another oxysterol, 25-hydroxycholesterol, but not by native free cholesterol. Addition of CD1d:IgG dimer protein or an anti-CD1d antibody, but not control IgG, significantly diminished 7K binding to CD1d-expressing HEK 293 cells. CD1d deficiency markedly diminished the 7K-binding in macrophages and smooth muscle cells. Western blot and gel shift assays demonstrated that CD1d-mediated 7K binding induced expression and activation of PPARγ. The PPARγ agonist PGJ2 enhances the 7K stimulatory effect on PPARγ expression and activity but the antagonist GW9662 inhibits the 7K effect on the CD1d-expressing cells., Conclusions: CD1d acts as a cell surface receptor that recognizes and binds oxysterols and initializes a pathway connecting oxysterol binding to PPARγ activation., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)