The N-Terminus of Human Sulfotransferase 2B1b─a Sterol-Sensing Allosteric Site.

Among human cytosolic sulfotransferases, SULT2B1b is highly specific for oxysterols─oxidized cholesterol derivatives, including nuclear-receptor ligands causally linked to skin and neurodegerative diseases, cancer and atherosclerosis. Sulfonation of signaling oxysterols redirects their receptor-binding functions, and controlling these functions is expected to prove valuable in disease prevention and treatment. SULT2B1b is distinct among the human SULT2 isoforms by virtue of its atypically long N-terminus, which extends 15 residues beyond the next longest N-terminus in the family. Here, in silico studies are used to predict that the N-terminal extension forms an allosteric pocket and to identify potential allosteres. One such allostere, quercetin, is used to confirm the existence of the pocket and to demonstrate that allostere binding inhibits turnover. The structure of the pocket is obtained by positioning quercetin on the enzyme, using spin-label-triangulation NMR, followed by NMR distance-constrained molecular dynamics docking. The model is confirmed using a combination of site-directed mutagenesis and initial-rate studies. Stopped-flow ligand-binding studies demonstrate that inhibition is achieved by stabilizing the closed form of the enzyme active-site cap, which encapsulates the nucleotide, slowing its release. Finally, endogenous oxysterols are shown to bind to the site in a highly selective fashion─one of the two immediate biosynthetic precursors of cholesterol (7-dehydrocholesterol) is an inhibitor, while the other (24-dehydrocholesterol) is not. These findings provide insights into the allosteric dialogue in which SULT2B1b participates in in vivo and establishes a template against which to develop isoform-specific inhibitors to control SULT2B1b biology.

Insights into estrogen receptor alpha modulation by cholestenoic acids.

Oxysterols are a family of over 25 cholesterol metabolites naturally produced by enzymatic or radical oxidation. They are involved in many physiological and pathological pathways. Although their activity has been mainly attributed to the modulation of the Liver X Receptors (LXR), it is currently accepted that oxysterols are quite promiscuous compounds, acting at several targets at the same time. The promiscuity of the oxysterols with the Estrogen Receptor α (ERα) is crucial in several pathologies such as ER+ breast cancer, inflammation and atherosclerosis. Regarding this matter, we have previously reported the synthesis, LXR activity and binding mode of a family of cholestenoic acid analogs with a modified side chain. Here we report the transcriptional activity on the ERα triggered by these compounds and details on the molecular determinants involved in their activities in order to establish structure-activity relationships to shed light over the molecular basis of the promiscuity of these compounds on ER/LXR responses. Our results show that 3ß-hydroxy-5-cholestenoic acid can interact with the ERα receptor in a way similar to 26-hydroxycholesterol and is an agonist of the receptor. Using molecular dynamics simulations, we were able to predict the ERα activity of a set of cholestenoic acid analogs with changes in the flexibility and/or steric requirements of the side chain, some of which exhibited selective activation of ERα or LXR.

Oxysterols Modify NLRP2 in Epithelial Cells, Identifying a Mediator of Ozone-induced Inflammation.

Ozone (O3) is a prevalent air pollutant causing lung inflammation. Previous studies demonstrate that O3 oxidizes lipids, such as cholesterol, in the airway to produce oxysterols, such as secosterol A (SecoA), which are electrophiles that are capable of forming covalent linkages preferentially with lysine residues and that consequently modify protein function. The breadth of proteins modified by this oxysterol as well as the biological consequences in the lung are unknown. By using an alkynyl-tagged form of SecoA and shotgun proteomics, we identified 135 proteins as being modified in bronchial epithelial cells. Among them was NLRP2 (NLR family pyrin domain-containing protein 2), which forms an alkynyl-tagged SecoA-protein adduct at lysine residue 1019 (K1019) in the terminal leucine-rich repeat region, a known regulatory region for NLR proteins. NLRP2 expression in airway epithelial cells was characterized, and CRISPR-Cas9 knockout (KO) and shRNA knockdown of NLRP2 were used to determine its function in O3-induced inflammation. No evidence for NLPR2 inflammasome formation or an NLRP2-dependent increase in caspase-1 activity in response to O3 was observed. O3-induced proinflammatory gene expression for CXCL2 and CXCL8/IL8 was further enhanced in NLRP2-KO cells, suggesting a negative regulatory role. Reconstitution of NLRP2-KO cells with the NLRP2 K1019 mutated to arginine partially blocked SecoA adduction and enhanced O3-induced IL-8 release as compared with wild-type NLRP2. Together, our findings uncover NLRP2 as a highly abundant, key component of proinflammatory signaling pathways in airway epithelial cells and as a novel mediator of O3-induced inflammation.

Identification of Anti-Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Oxysterol Derivatives In Vitro.

The development of effective antiviral drugs targeting the severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) is urgently needed to combat the coronavirus disease 2019 (COVID-19). We have previously studied the use of semi-synthetic derivatives of oxysterols, oxidized derivatives of cholesterol as drug candidates for the inhibition of cancer, fibrosis, and bone regeneration. In this study, we screened a panel of naturally occurring and semi-synthetic oxysterols for anti-SARS-CoV-2 activity using a cell culture infection assay. We show that the natural oxysterols, 7-ketocholesterol, 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 27-hydroxycholesterol, substantially inhibited SARS-CoV-2 propagation in cultured cells. Among semi-synthetic oxysterols, Oxy210 and Oxy232 displayed more robust anti-SARS-CoV-2 activities, reducing viral replication more than 90% at 10 µM and 99% at 15 µM, respectively. When orally administered in mice, peak plasma concentrations of Oxy210 fell into a therapeutically relevant range (19 µM), based on the dose-dependent curve for antiviral activity in our cell-based assay. Mechanistic studies suggest that Oxy210 reduced replication of SARS-CoV-2 by disrupting the formation of double-membrane vesicles (DMVs); intracellular membrane compartments associated with viral replication. Our study warrants further evaluation of Oxy210 and Oxy232 as a safe and reliable oral medication, which could help protect vulnerable populations with increased risk of developing COVID-19.

Electrophilic oxysterols: generation, measurement and protein modification.

Cholesterol is an essential component of mammalian plasma membranes. Alterations in sterol metabolism or oxidation have been linked to various pathological conditions, including cardiovascular diseases, cancer, and neurodegenerative disorders. Unsaturated sterols are vulnerable to oxidation induced by singlet oxygen and other reactive oxygen species. This process yields reactive sterol oxidation products, including hydroperoxides, epoxides as well as aldehydes. These oxysterols, in particular those with high electrophilicity, can modify nucleophilic sites in biomolecules and affect many cellular functions. Here, we review the generation and measurement of reactive sterol oxidation products with emphasis on cholesterol hydroperoxides and aldehyde derivatives (electrophilic oxysterols) and their effects on protein modifications.

A Comprehensive Review on Oxysterols and Related Diseases.

The present review aims to provide a complete and comprehensive summary of current literature relevant to oxysterols and related diseases. Oxidation of cholesterol leads to the formation of a large number of oxidized products, generally known as oxysterols. They are intermediates in the biosynthesis of bile acids, steroid hormones, and 1,25- dihydroxyvitamin D3. Although oxysterols are considered as metabolic intermediates, there is a growing body of evidence that many of them are bioactive, and their absence or excess may be part of the cause of a disease phenotype. These compounds derive from either enzymatic or non-enzymatic oxidation of cholesterol. This study provides comprehensive information about the structures, formation, and types of oxysterols even when involved in certain disease states, focusing on their effects on metabolism and linkages with these diseases. The role of specific oxysterols as mediators in various disorders, such as degenerative (age-related) and cancer-related disorders, has now become clearer. Oxysterol levels may be employed as suitable markers for the diagnosis of specific diseases or in predicting the incidence rate of diseases, such as diabetes mellitus, Alzheimer's disease, multiple sclerosis, osteoporosis, lung cancer, breast cancer, and infertility. However, further investigations may be required to confirm these mentioned possibilities.

Oxysterols provide innate immunity to bacterial infection by mobilizing cell surface accessible cholesterol.

Cholesterol 25-hydroxylase (CH25H) is an interferon-stimulated gene that converts cholesterol to the oxysterol 25-hydroxycholesterol (25HC). Circulating 25HC modulates essential immunological processes including antiviral immunity, inflammasome activation and antibody class switching; and dysregulation of CH25H may contribute to chronic inflammatory disease and cancer. Although 25HC is a potent regulator of cholesterol storage, uptake, efflux and biosynthesis, how these metabolic activities reprogram the immunological state of target cells remains poorly understood. Here, we used recently designed toxin-based biosensors that discriminate between distinct pools of plasma membrane cholesterol to elucidate how 25HC prevents Listeria monocytogenes from traversing the plasma membrane of infected host cells. The 25HC-mediated activation of acyl-CoA:cholesterol acyltransferase (ACAT) triggered rapid internalization of a biochemically defined fraction of cholesterol, termed 'accessible' cholesterol, from the plasma membrane while having little effect on cholesterol in complexes with sphingomyelin. We show that evolutionarily distinct bacterial species, L. monocytogenes and Shigella flexneri, exploit the accessible pool of cholesterol for infection and that acute mobilization of this pool by oxysterols confers immunity to these pathogens. The significance of this signal-mediated membrane remodelling pathway probably extends beyond host defence systems, as several other biologically active oxysterols also mobilize accessible cholesterol through an ACAT-dependent mechanism.

Cryo-EM structure of oxysterol-bound human Smoothened coupled to a heterotrimeric Gi.

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway1,2. It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins3. Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24(S),25-epoxycholesterol and coupled to a heterotrimeric Gi protein. The structure reveals a ligand-binding site for 24(S),25-epoxycholesterol in the 7-transmembrane region, as well as a Gi-coupled activation mechanism of human SMO. Notably, the Gi protein presents a different arrangement from that of class-A GPCR-Gi complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR.

Inhibition of Hedgehog Signaling in Fibroblasts, Pancreatic, and Lung Tumor Cells by Oxy186, an Oxysterol Analogue with Drug-Like Properties.

The widespread involvement of the Hedgehog (Hh) signaling pathway in human malignancies has motivated the clinical development of Smoothened (Smo) antagonists, such as vismodegib and sonidegib. However, Smo antagonists have failed to benefit patients suffering from Hh pathway-dependent solid tumors, such as pancreatic, colorectal, or ovarian cancer. Hh-dependent cancers are often driven by activating mutations that occur downstream of Smo and directly activate the transcription factors known as glioma-associated oncogenes (Gli1-3). Hence, the direct targeting of Gli could be a more effective strategy for achieving disease modification compared to Smo antagonism. In this study, we report on the biological and pharmacological evaluation of Oxy186, a semisynthetic oxysterol analogue, as a novel inhibitor of Hh signaling acting downstream of Smo, with encouraging drug-like properties. Oxy186 exhibits strong inhibition of ligand-induced Hh signaling in NIH3T3-E1 fibroblasts, as well as in constitutively activated Hh signaling in Suppressor of Fused (Sufu) null mouse embryonic fibroblast (MEF) cells. Oxy186 also inhibits Gli1 transcriptional activity in NIH3T3-E1 cells expressing exogenous Gli1 and Gli-dependent reporter constructs. Furthermore, Oxy186 suppresses Hh signaling in PANC-1 cells, a human pancreatic ductal adenocarcinoma (PDAC) tumor cell line, as well as PANC-1 cell proliferation in vitro, and in human lung cancer cell lines, A549 and H2039.

Omics analysis of oxysterols to better understand their pathophysiological role.

High amounts of cholesterol have been definitely associated with the pathogenesis of several diseases, including metabolic and neurodegenerative disorders, cardiovascular diseases, and cancer. In all these pathologies the exacerbation of pro-oxidant and inflammatory responses is a consistent feature. In this scenario, species derived from enzymatic and non-enzymatic cholesterol oxidation, namely oxysterols, are strongly suspected to play a primary role. The consideration of these bioactive lipids is therefore helpful in investigating pathological mechanisms and may also acquire clinical value for the diagnosis and treatment of diseases. For this purpose and considering that a great number of oxysterols may be present together in the body, the employment of lipidomics technology certainly represents a powerful strategy for the simultaneous detection and characterization of these compounds in biological specimens. In this review, we will discuss the applicability of the lipidomics approach in the study of the association between oxysterols and diseases.

Fast liquid chromatography-mass spectrometry reveals side chain oxysterol heterogeneity in breast cancer tumour samples.

Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system ("AFFL"). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.

Oxysterols from a Marine Sponge Inflatella sp. and Their Action in 6-Hydroxydopamine-Induced Cell Model of Parkinson's Disease.

Four new oxysterols 1⁻4 along with previously known oxygenated sterols 5⁻14 were isolated from the sponge Inflatella sp., collected from the Sea of Okhotsk. Structures of 1⁻4 were elucidated by the detailed NMR spectroscopic and mass-spectrometric analyses as well as by comparison of the corresponding experimental data with those reported in literature. The influence of compounds 1⁻14 on the viability of neuronal Neuro2a cells treated by 6-hydroxydopamine and reactive oxygen species (ROS) formation in these cells was investigated.

7-Ketocholesterol enhances leukocyte adhesion to endothelial cells via p38MAPK pathway.

7-Ketocholesterol is a major dietary cholesterol oxidation product found in high concentrations in atherosclerotic plaques, which contribute to the development of atherosclerosis. This study aimed to investigate the effects of 7-ketocholesterol on endothelial inflammation, as well as the underlying mechanisms. Pretreatment of human umbilical vein endothelial cells (HUVEC) with 7-ketocholesterol significantly enhanced the total interactions between human monocytic cells (THP-1 cell line) and TNFα-activated HUVECs under physiological flow conditions, compared to pretreatment with cholesterol (TNFα+50 µM cholesterol: 13.1 ± 0.54 cells/CPF, TNFα+50 µM 7-ketocholesterol: 18.9 ± 0.35 cells/CPF, p < 0.01). 7-Ketocholesterol enhanced the expression of E-selectin, ICAM-1, and VCAM-1 proteins. It also activated p38 mitogen-activated protein kinase (MAPK), and treatment with a p38 MAPK inhibitor inhibited both E-selectin expression via ATF-2 activation and 7-ketocholesterol-induced THP-1 adhesion to HUVECs. These findings suggest that 7-ketocholesterol enhances leukocyte-endothelial interactions by upregulating the expression of adhesion molecules, presumably via the p38 MAPK-dependent pathway.

Myeloid apolipoprotein E controls dendritic cell antigen presentation and T cell activation.

Cholesterol homeostasis has a pivotal function in regulating immune cells. Here we show that apolipoprotein E (apoE) deficiency leads to the accumulation of cholesterol in the cell membrane of dendritic cells (DC), resulting in enhanced MHC-II-dependent antigen presentation and CD4+ T-cell activation. Results from WT and apoE KO bone marrow chimera suggest that apoE from cells of hematopoietic origin has immunomodulatory functions, regardless of the onset of hypercholesterolemia. Humans expressing apoE4 isoform (ε4/3-ε4/4) have increased circulating levels of activated T cells compared to those expressing WT apoE3 (ε3/3) or apoE2 isoform (ε2/3-ε2/2). This increase is caused by enhanced antigen-presentation by apoE4-expressing DCs, and is reversed when these DCs are incubated with serum containing WT apoE3. In summary, our study identifies myeloid-produced apoE as a key physiological modulator of DC antigen presentation function, paving the way for further explorations of apoE as a tool to improve the management of immune diseases.

Identification of unusual oxysterols and bile acids with 7-oxo or 3ß,5α,6ß-trihydroxy functions in human plasma by charge-tagging mass spectrometry with multistage fragmentation.

7-Oxocholesterol (7-OC), 5,6-epoxycholesterol (5,6-EC), and its hydrolysis product cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol) are normally minor oxysterols in human samples; however, in disease, their levels may be greatly elevated. This is the case in plasma from patients suffering from some lysosomal storage disorders, e.g., Niemann-Pick disease type C, or the inborn errors of sterol metabolism, e.g., Smith-Lemli-Opitz syndrome and cerebrotendinous xanthomatosis. A complication in the analysis of 7-OC and 5,6-EC is that they can also be formed ex vivo from cholesterol during sample handling in air, causing confusion with molecules formed in vivo. When formed endogenously, 7-OC, 5,6-EC, and 3ß,5α,6ß-triol can be converted to bile acids. Here, we describe methodology based on chemical derivatization and LC/MS with multistage fragmentation (MSn) to identify the necessary intermediates in the conversion of 7-OC to 3ß-hydroxy-7-oxochol-5-enoic acid and 5,6-EC and 3ß,5α,6ß-triol to 3ß,5α,6ß-trihydroxycholanoic acid. Identification of intermediate metabolites is facilitated by their unusual MSn fragmentation patterns. Semiquantitative measurements are possible, but absolute values await the synthesis of isotope-labeled standards.

Cytotoxic activity of alkyl benzoate and fatty acids from the red sea sponge Hyrtios erectus.

The chemical investigation of the methylene chloride fraction of marine sponge Hyrtios erectus led to the isolation of the known oxysterol (2) along with a new alkyl benzoate compound identified by spectroscopic methods (NMR and MS) as 4'-methylheptyl benzoate (1), whilst the n-butanol fraction afforded the known indole 3-carbaldehyde and ß-carboline derivatives. Moreover, the hexane fraction was analysed by GC-MS for their fatty acids (FAs). A total of 17 FAs with chain lengths between 14 and 25 carbons were identified. Methyl-branched FAs are predominated suggesting the presence of bacterial symbionts in the H. erectus sponge. Furthermore, compounds 1 and 2 displayed significant cytotoxicity against breast adenocarcinoma (MCF-7) with IC50 values of 2.4 and 3.8 µM, respectively, since compound 2 was also shown to have potent cytotoxic effect against hepatocellular carcinoma cells (HepG 2) with IC50 value of 1.3 µM.

Design and Characterization of a Therapeutic Non-phospholipid Liposomal Nanocarrier with Osteoinductive Characteristics To Promote Bone Formation.

Sterosomes are recently developed types of non-phospholipid liposomes formed from single-chain amphiphiles and high content of sterols. Although sterosomes presented significantly increased stability compared to conventional phospholipid liposomes, current sterosome biomaterials are not truly bioactive and have no intrinsic therapeutic effects. The purpose of this study was to develop a sterosome formulation with osteoinductive properties by an effective selection of sterol, one of the sterosome components. Oxysterols are oxidized derivatives of cholesterol and are known to stimulate osteogenesis and bone formation. Thus, 20S-hydroxycholesterol (Oxy), one of the most potent oxysterols for bone regeneration, was examined as a promising candidate molecule to form fluid lamellar phases with a single-chain amphiphile, namely, stearylamine (SA). First, the optimal composition was identified by investigating the phase behavior of SA/Oxy mixtures. Next, the capacity of the optimized SA/Oxy sterosomes to promote osteogenic differentiation of bone marrow stromal cells was assessed in vitro in a hydrogel environment. Furthermore, we explored the effects of osteogenic oxysterol sterosomes in vivo with the mouse critical-sized calvarial defect model. Our results showed that SA/Oxy sterosomes induced osteogenic differentiation in vitro and enhanced calvarial healing without delivery of additional therapeutic agents, indicating their intrinsic bone-forming potential. This study suggests a promising non-phospholipid liposomal platform with osteoinductive properties for delivery of small molecular drugs and/or other therapeutic genes for enhanced bone formation.

Oxysterols and mesenchymal stem cell biology.

Oxysterols are oxidized products of cholesterol that play several roles in various pathophysiological processes, including the control of lipid metabolism, immunological processes, and cytotoxicity. Mesenchymal stem cells are multipotent cells with properties of self-renewal and the ability to differentiate into other cell types, including osteoblasts and adipocytes. Here, we review the literature regarding the effects of oxysterols on mesenchymal stem cell differentiation and the main signaling pathways involved in this process.

Hedgehog Signaling: From Basic Biology to Cancer Therapy.

The Hedgehog (HH) signaling pathway was discovered originally as a key pathway in embryonic patterning and development. Since its discovery, it has become increasingly clear that the HH pathway also plays important roles in a multitude of cancers. Therefore, HH signaling has emerged as a therapeutic target of interest for cancer therapy. In this review, we provide a brief overview of HH signaling and the key molecular players involved and offer an up-to-date summary of our current knowledge of endogenous and exogenous small molecules that modulate HH signaling. We discuss experiences and lessons learned from the decades-long efforts toward the development of cancer therapies targeting the HH pathway. Challenges to develop next-generation cancer therapies are highlighted.