Inhibition of post-lanosterol biosynthesis by fentanyl: potential implications for Fetal Fentanyl Syndrome (FFS).

A recent study discovered a novel, complex developmental disability syndrome, most likely caused by maternal fentanyl use disorder. This Fetal Fentanyl Syndrome (FFS) is biochemically characterized by elevated 7-dehydrocholesterol (7-DHC) levels in neonates, raising the question if fentanyl inhibition of the dehydrocholesterol reductase 7 (DHCR7) enzyme is causal for the emergence of the pathophysiology and phenotypic features of FFS. To test this hypothesis, we undertook a series of experiments on Neuro2a cells, primary mouse neuronal and astrocytic cultures, and human dermal fibroblasts (HDFs) with DHCR7+/+ and DHCR7+/- genotype. Our results revealed that in vitro exposure to fentanyl disrupted sterol biosynthesis across all four in vitro models. The sterol biosynthesis disruption by fentanyl was complex, and encompassed the majority of post-lanosterol intermediates, including elevated 7-DHC and decreased desmosterol (DES) levels across all investigated models. The overall findings suggested that maternal fentanyl use in the context of an opioid use disorder leads to FFS in the developing fetus through a strong disruption of the whole post-lanosterol pathway that is more complex than a simple DHCR7 inhibition. In follow-up experiments we found that heterozygous DHCR7+/- HDFs were significantly more susceptible to the sterol biosynthesis inhibitory effects of fentanyl than wild-type DHCR7+/+ fibroblasts. These data suggest that DHCR7+/- heterozygosity of mother and/or developing child (and potentially other sterol biosynthesis genes), when combined with maternal fentanyl use disorder, might be a significant contributory factor to the emergence of FFS in the exposed offspring. In a broader context, we believe that evaluation of new and existing medications for their effects on sterol biosynthesis should be an essential consideration during drug safety determinations, especially in pregnancy.

PRMT5 in T Cells Drives Th17 Responses, Mixed Granulocytic Inflammation, and Severe Allergic Airway Inflammation.

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4+ T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt's agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma.

Effects of Neonatal Hypoxic-Ischemic Injury on Brain Sterol Synthesis and Metabolism.

BACKGROUND: Neonatal hypoxic-ischemic brain injury (HIBI) results from disruptions to blood supply and oxygen in the perinatal brain. The goal of this study was to measure brain sterol metabolites and plasma oxysterols after injury in a neonatal HIBI mouse model to assess for potential therapeutic targets in the brain biochemistry as well as potential circulating diagnostic biomarkers. METHODS: Postnatal day 9 CD1-IGS mouse pups were randomized to HIBI induced by carotid artery ligation followed by 30 minutes at 8% oxygen or to sham surgery and normoxia. Brain tissue was collected for sterol analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plasma was collected for oxysterol analysis by LC-MS/MS. RESULTS: There were minimal changes in brain sterol concentrations in the first 72 hours after HIBI. In severely injured brains, there was a significant increase in desmosterol, 7-DHC, 8-DHC, and cholesterol 24 hours after injury in the ipsilateral tissue. Lanosterol, 24-dehydrolathosterol, and 14-dehydrozymostenol decreased in plasma 24 hours after injury. Severe neonatal HIBI was associated with increased cholesterol and sterol precursors in the cortex at 24 hours after injury. CONCLUSIONS: Differences in plasma oxysterols were seen at 24 hours but were not present at 30 minutes after injury, suggesting that these sterol intermediates would be of little value as early diagnostic biomarkers.

Sterols and immune mechanisms in asthma.

The field of sterol and oxysterol biology in lung disease has recently gained attention, revealing a unique need for sterol uptake and metabolism in the lung. The presence of cholesterol transport, biosynthesis, and sterol/oxysterol-mediated signaling in immune cells suggests a role in immune regulation. In support of this idea, statin drugs that inhibit the cholesterol biosynthesis rate-limiting step enzyme, hydroxymethyl glutaryl coenzyme A reductase, show immunomodulatory activity in several models of inflammation. Studies in human asthma reveal contradicting results, whereas promising retrospective studies suggest benefits of statins in severe asthma. Here, we provide a timely review by discussing the role of sterols in immune responses in asthma, analytical tools to evaluate the role of sterols in disease, and potential mechanistic pathways and targets relevant to asthma. Our review reveals the importance of sterols in immune processes and highlights the need for further research to solve critical gaps in the field.

Vaping Induced Cannabidiol (CBD) Oxidation Product CBD Quinone Forms Protein Adducts with KEAP1 and Activates KEAP1-Nrf2 Genes.

Cannabidiol (CBD) vaping products have become widely available in the U.S. since their legalization in 2018. However, little is known about their respiratory health effects. Here we show that aerosolization of commercial CBD vaping products generates a reactive CBD quinone (CBDQ) which forms adducts with protein cysteine residues. Using click chemistry and a novel in vitro vaping product exposure system (VaPES), we further demonstrate that CBDQ forms adducts with human bronchial epithelial cell proteins including Keap1 and activates KEAP1-Nrf2 stress response pathway genes. These results suggest that vaping CBD alters protein function and induces cellular stress pathways in the lung.

Metabolic Control of Sensory Neuron Survival by the p75 Neurotrophin Receptor in Schwann Cells.

We report that the neurotrophin receptor p75 contributes to sensory neuron survival through the regulation of cholesterol metabolism in Schwann cells. Selective deletion of p75 in mouse Schwann cells of either sex resulted in a 30% loss of dorsal root ganglia (DRG) neurons and diminished thermal sensitivity. P75 regulates Schwann cell cholesterol biosynthesis in response to BDNF, forming a co-receptor complex with ErbB2 and activating ErbB2-mediated stimulation of sterol regulatory element binding protein 2 (SREBP2), a master regulator of cholesterol synthesis. Schwann cells lacking p75 exhibited decreased activation of SREBP2 and a reduction in 7-dehydrocholesterol (7-DHC) reductase (DHCR7) expression, resulting in accumulation of the neurotoxic intermediate, 7-dehyrocholesterol in the sciatic nerve. Restoration of DHCR7 in p75 null Schwann cells in mice significantly attenuated DRG neuron loss. Together, these results reveal a mechanism by which the disruption of lipid metabolism in glial cells negatively influences sensory neuron survival, which has implications for a wide range of peripheral neuropathies.SIGNIFICANCE STATEMENT Although expressed in Schwann cells, the role of p75 in myelination has remained unresolved in part because of its dual expression in sensory neurons that Schwann cells myelinate. When p75 was deleted selectively among Schwann cells, myelination was minimally affected, while sensory neuron survival was reduced by 30%. The phenotype is mainly due to dysregulation of cholesterol biosynthesis in p75-deficient Schwann cells, leading to an accumulation of neurotoxic cholesterol precursor, 7-dehydrocholesterol (7-DHC). Mechanism-wise, we discovered that in response to BDNF, p75 recruits and activates ErbB2 independently of ErbB3, thereby stimulating the master regulator, sterol regulatory element binding protein 2 (SREBP2). These results together highlight a novel role of p75 in Schwann cells in regulating DRG neuron survival by orchestrating proper cholesterol metabolism.

Glucocorticoids are induced while dihydrotestosterone levels are suppressed in 5-alpha reductase inhibitor treated human benign prostate hyperplasia patients.

BACKGROUND: The development of benign prostatic hyperplasia (BPH) and medication-refractory lower urinary tract symptoms (LUTS) remain poorly understood. This study attempted to characterize the pathways associated with failure of medical therapy for BPH/LUTS. METHODS: Transitional zone tissue levels of cholesterol and steroids were measured in patients who failed medical therapy for BPH/LUTS and controls. Prostatic gene expression was measured using qPCR and BPH cells were used in organoid culture to study prostatic branching. RESULTS: BPH patients on 5-α-reductase inhibitor (5ARI) showed low levels of tissue dihydrotestosterone (DHT), increased levels of steroid 5-α-reductase type II (SRD5A2), and diminished levels of androgen receptor (AR) target genes, prostate-specific antigen (PSA), and transmembrane serine protease 2 (TMPRSS2). 5ARI raised prostatic tissue levels of glucocorticoids (GC), whereas alpha-adrenergic receptor antagonists (α-blockers) did not. Nuclear localization of GR in prostatic epithelium and stroma appeared in all patient samples. Treatment of four BPH organoid cell lines with dexamethasone, a synthetic GC, resulted in budding and branching. CONCLUSIONS: After failure of medical therapy for BPH/LUTS, 5ARI therapy continued to inhibit androgenesis but a 5ARI-induced pathway increased tissue levels of GC not seen in patients on α-blockers. GC stimulation of organoids indicated that the GC receptors are a trigger for controlling growth of prostate glands. A 5ARI-induced pathway revealed GC activation can serve as a master regulator of prostatic branching and growth.

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.

Generation and validation of a conditional knockout mouse model for the study of the Smith-Lemli-Opitz syndrome.

Smith-Lemli-Opitz Syndrome (SLOS) is a developmental disorder (OMIM #270400) caused by autosomal recessive mutations in the Dhcr7 gene, which encodes the enzyme 3ß-hydroxysterol-Δ7 reductase. SLOS patients present clinically with dysmorphology and neurological, behavioral, and cognitive defects, with characteristically elevated levels of 7-dehydrocholesterol (7-DHC) in all bodily tissues and fluids. Previous mouse models of SLOS have been hampered by postnatal lethality when Dhcr7 is knocked out globally, while a hypomorphic mouse model showed improvement in the biochemical phenotype with aging and did not manifest most other characteristic features of SLOS. We report the generation of a conditional knockout of Dhcr7 (Dhcr7flx/flx), validated by generating a mouse with a liver-specific deletion (Dhcr7L-KO). Phenotypic characterization of liver-specific knockout mice revealed no significant changes in viability, fertility, growth curves, liver architecture, hepatic triglyceride secretion, or parameters of systemic glucose homeostasis. Furthermore, qPCR and RNA-Seq analyses of livers revealed no perturbations in pathways responsible for cholesterol synthesis, either in male or in female Dhcr7L-KO mice, suggesting that hepatic disruption of postsqualene cholesterol synthesis leads to minimal impact on sterol metabolism in the liver. This validated conditional Dhcr7 knockout model may now allow us to systematically explore the pathophysiology of SLOS, by allowing for temporal, cell and tissue-specific loss of DHCR7.

Ozone-derived oxysterols impair lung macrophage phagocytosis via adduction of some phagocytosis receptors.

Inhalation of the ambient air pollutant ozone causes lung inflammation and can suppress host defense mechanisms, including impairing macrophage phagocytosis. Ozone reacts with cholesterol in the lung to form oxysterols, like secosterol A and secosterol B (SecoA and SecoB), which can form covalent adducts on cellular proteins. How oxysterol-protein adduction modifies the function of lung macrophages is unknown. Herein, we used a proteomic screen to identify lung macrophage proteins that form adducts with ozone-derived oxysterols. Functional ontology analysis of the adductome indicated that protein binding was a major function of adducted proteins. Further analysis of specific proteins forming adducts with SecoA identified the phagocytic receptors CD206 and CD64. Adduction of these receptors with ozone-derived oxysterols impaired ligand binding and corresponded with reduced macrophage phagocytosis. This work suggests a novel mechanism for the suppression of macrophage phagocytosis following ozone exposure through the generation of oxysterols and the formation of oxysterol-protein adducts on phagocytic receptors.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Maternal cariprazine exposure inhibits embryonic and postnatal brain cholesterol biosynthesis.

Cariprazine (CAR) is a strong inhibitor of the Dhcr7 enzyme, the last enzyme in the cholesterol biosynthesis pathway. We assessed the effects of CAR on maternally exposed Dhcr7+/- and wild-type mouse offspring, and tested the biochemical effects of CAR in human serum samples. Dhcr7+/- and wild-type time-pregnant mice were exposed to vehicle or 0.2 mg/kg CAR from E12 to E19. Levels of CAR, CAR metabolites, sterols, and oxysterols were measured in the brain of maternally exposed offspring at various time points using LC-MS/MS. Embryonic exposure to CAR significantly increased levels of 7-DHC in all organs of exposed embryos, with a particularly strong effect in the brain. Detectable levels of CAR and elevated 7-DHC were observed in the brain of newborn pups 14 days after drug exposure. In addition, CAR altered sterol metabolism in all animals analyzed, with the strongest effect on the brain of Dhcr7+/- pups born to Dhcr7+/- dams. Furthermore, CAR elevated toxic oxysterols in the brain of maternally exposed Dhcr7+/- offspring to levels approaching those seen in a mouse model of Smith-Lemli-Opitz syndrome. Finally, we observed that patients taking CAR have elevated 7-DHC in their serum. In summary, maternal DHCR7 heterozygosity, combined with offspring DHCR7 heterozygosity might represent a vulnerability factor to medications that interfere with sterol biosynthesis. Due to the conserved sterol biosynthesis between mice and humans, we suggest that the 1-3% of patient population with single-allele DHCR7 mutations might not be ideal candidates for CAR use, especially if they are nursing, pregnant or plan to become pregnant.

Maternal aripiprazole exposure interacts with 7-dehydrocholesterol reductase mutations and alters embryonic neurodevelopment.

Mutations in both copies in the gene encoding 7-dehydrocholesterol reductase (DHCR7) cause Smith-Lemli-Opitz Syndrome (SLOS), which is characterized by a toxic elevation in 7-dehydrocholesterol (7-DHC). Aripiprazole (ARI) exposure, independent of genetic mutations, also leads to elevation of 7-DHC. We investigated the combined effect of a single-copy Dhcr7+/- mutation and maternal ARI exposure on the developing offspring brain. We generated a time-pregnant mouse model where WT and Dhcr7+/- embryos were maternally exposed to ARI or vehicle (VEH) from E12 to E19 (5 mg/kg). Levels of cholesterol, its precursors, ARI and its metabolites were measured at P0. We found that ARI and its metabolites were transported across the placenta and reached the brain of offspring. Maternal ARI exposure led to decreased viability of embryos and increased 7-DHC levels, regardless of maternal or offspring Dhcr7 genotype. In addition, Dhcr7+/- pups were more vulnerable to maternal ARI exposure than their WT littermates, and maternal Dhcr7+/- genotype also exacerbated offspring response to ARI treatment. Finally, both 7-DHC levels and 7-DHC/cholesterol ratio is the highest in Dhcr7+/- pups from Dhcr7+/- mothers exposed to ARI, underscoring a potentially dangerous interaction between maternal genotype×embryonic genotype×treatment. Our findings have important clinical implications. SLOS patients should avoid drugs that increase 7-DHC levels such as ARI, trazodone and haloperidol. In addition, treatment with 7-DHC elevating substances might be potentially unsafe for the 1-1.5% of population with single-allele disruptions of the DHCR7 gene. Finally, prenatal and parental genetic testing for DHCR7 should be considered before prescribing sterol-interfering medications during pregnancy.

Evaluating a targeted multiple reaction monitoring approach to global untargeted lipidomic analyses of human plasma.

RATIONALE: The Lipidyzer platform was recently updated on a SCIEX QTRAP 6500+ mass spectrometer and offers a targeted lipidomics assay including 1150 different lipids. We evaluated this targeted approach using human plasma samples and compared the results against a global untargeted lipidomics method using a high-resolution Q Exactive HF Orbitrap mass spectrometer. METHODS: Lipids from human plasma samples (N = 5) were extracted using a modified Bligh-Dyer approach. A global untargeted analysis was performed using a Thermo Orbitrap Q Exactive HF mass spectrometer, followed by data analysis using Progenesis QI software. Multiple reaction monitoring (MRM)-based targeted analysis was performed using a QTRAP 6500+ mass spectrometer, followed by data analysis using SCIEX OS software. The samples were injected on three separate days to assess reproducibility for both approaches. RESULTS: Overall, 465 lipids were identified from 11 lipid classes in both approaches, of which 159 were similar between the methods, 168 lipids were unique to the MRM approach, and 138 lipids were unique to the untargeted approach. Phosphatidylcholine and phosphatidylethanolamine species were the most commonly identified using the untargeted approach, while triacylglycerol species were the most commonly identified using the targeted MRM approach. The targeted MRM approach had more consistent relative abundances across the three days than the untargeted approach. Overall, the coefficient of variation for inter-day comparisons across all lipid classes was ∼ 23% for the untargeted approach and ∼ 9% for the targeted MRM approach. CONCLUSIONS: The targeted MRM approach identified similar numbers of lipids to a conventional untargeted approach, but had better representation of 11 lipid classes commonly identified by both approaches. Based on the separation methods employed, the conventional untargeted approach could better detect phosphatidylcholine and sphingomyelin lipid classes. The targeted MRM approach had lower inter-day variability than the untargeted approach when tested using a small group of plasma samples. These studies highlight the advantages in using targeted MRM approaches for human plasma lipidomics analysis.

Small Molecule Antipsychotic Aripiprazole Potentiates Ozone-Induced Inflammation in Airway Epithelium.

Inhaled ground level ozone (O3) has well described adverse health effects, which may be augmented in susceptible populations. While conditions, such as pre-existing respiratory disease, have been identified as factors enhancing susceptibility to O3-induced health effects, the potential for chemical interactions in the lung to sensitize populations to pollutant-induced responses has not yet been studied. In the airways, inhaled O3 reacts with lipids, such as cholesterol, to generate reactive and electrophilic oxysterol species, capable of causing cellular dysfunction and inflammation. The enzyme regulating the final step of cholesterol biosynthesis, 7-dehydrocholesterol reductase (DHCR7), converts 7-dehydrocholesterol (7-DHC) to cholesterol. Inhibition of DHCR7 increases the levels of 7-DHC, which is much more susceptible to oxidation than cholesterol. Chemical analysis established the capacity for a variety of small molecule antipsychotic drugs, like Aripiprazole (APZ), to inhibit DHCR7 and elevate circulating 7-DHC. Our results show that APZ and the known DHCR7 inhibitor, AY9944, increase 7-DHC levels in airway epithelial cells and potentiate O3-induced IL-6 and IL-8 expression and cytokine release. Targeted immune-related gene array analysis demonstrates that APZ significantly modified O3-induced expression of 16 genes, causing dysregulation in expression of genes associated with leukocyte recruitment and inflammatory response. Additionally, we find that APZ increases O3-induced IL-6 and IL-8 expression in human nasal epithelial cells from male but not female donors. Overall, the evidence we provide describes a novel molecular mechanism by which chemicals, such as APZ, that perturb cholesterol biosynthesis affect O3-induced biological responses.

Desmosterolosis and desmosterol homeostasis in the developing mouse brain.

Cholesterol serves as a building material for cellular membranes and plays an important role in cellular metabolism. The brain relies on its own cholesterol biosynthesis, which starts during embryonic development. Cholesterol is synthesized from two immediate precursors, desmosterol and 7-dehydrocholesterol (7-DHC). Mutations in the DHCR24 enzyme, which converts desmosterol into cholesterol, lead to desmosterolosis, an autosomal recessive developmental disorder. In this study, we assessed the brain content of desmosterol, 7-DHC, and cholesterol from development to adulthood, and analyzed the biochemical, molecular, and anatomical consequences of Dhcr24 mutations on the sterol profile in a mouse model of desmosterolosis and heterozygous Dhcr24+/- carriers. Our HPLC-MS/MS studies revealed that by P0 desmosterol almost entirely replaced cholesterol in the Dhcr24-KO brain. The greatly elevated desmosterol levels were also present in the Dhcr24-Het brains irrespective of maternal genotype, persisting into adulthood. Furthermore, Dhcr24-KO mice brains showed complex changes in expression of lipid and sterol transcripts, nuclear receptors, and synaptic plasticity transcripts. Cultured Dhcr24-KO neurons showed increased arborization, which was also present in the Dhcr24-KO mouse brains. Finally, we observed a shared pathophysiological mechanism between the mouse models of desmosterolosis and Smith-Lemli-Opitz syndrome (a genetic disorder of conversion of 7-DHC to cholesterol).

Chemoproteomics Reveals Chemical Diversity and Dynamics of 4-Oxo-2-nonenal Modifications in Cells.

4-Oxo-2-nonenal (ONE) derived from lipid peroxidation modifies nucleophiles and transduces redox signaling by its reactions with proteins. However, the molecular interactions between ONE and complex proteomes and their dynamics in situ remain largely unknown. Here we describe a quantitative chemoproteomic analysis of protein adduction by ONE in cells, in which the cellular target profile of ONE is mimicked by its alkynyl surrogate. The analyses reveal four types of ONE-derived modifications in cells, including ketoamide and Schiff-base adducts to lysine, Michael adducts to cysteine, and a novel pyrrole adduct to cysteine. ONE-derived adducts co-localize and exhibit crosstalk with many histone marks and redox sensitive sites. All four types of modifications derived from ONE can be reversed site-specifically in cells. Taken together, our study provides much-needed mechanistic insights into the cellular signaling and potential toxicities associated with this important lipid derived electrophile.

Systematic and Quantitative Assessment of Hydrogen Peroxide Reactivity With Cysteines Across Human Proteomes.

Protein cysteinyl residues are the mediators of hydrogen peroxide (H2O2)-dependent redox signaling. However, site-specific mapping of the selectivity and dynamics of these redox reactions in cells poses a major analytical challenge. Here we describe a chemoproteomic platform to systematically and quantitatively analyze the reactivity of thousands of cysteines toward H2O2 in human cells. We identified >900 H2O2-sensitive cysteines, which are defined as the H2O2-dependent redoxome. Although redox sites associated with antioxidative and metabolic functions are consistent, most of the H2O2-dependent redoxome varies dramatically between different cells. Structural analyses reveal that H2O2-sensitive cysteines are less conserved than their redox-insensitive counterparts and display distinct sequence motifs, structural features, and potential for crosstalk with lysine modifications. Notably, our chemoproteomic platform also provides an opportunity to predict oxidation-triggered protein conformational changes. The data are freely accessible as a resource at http://redox.ncpsb.org/OXID/.

Endogenous B-ring oxysterols inhibit the Hedgehog component Smoothened in a manner distinct from cyclopamine or side-chain oxysterols.

Cellular lipids are speculated to act as key intermediates in Hedgehog signal transduction, but their precise identity and function remain enigmatic. In an effort to identify such lipids, we pursued a Hedgehog pathway inhibitory activity that is particularly abundant in flagellar lipids of Chlamydomonas reinhardtii, resulting in the purification and identification of ergosterol endoperoxide, a B-ring oxysterol. A mammalian analog of ergosterol, 7-dehydrocholesterol (7-DHC), accumulates in Smith-Lemli-Opitz syndrome, a human genetic disease that phenocopies deficient Hedgehog signaling and is caused by genetic loss of 7-DHC reductase. We found that depleting endogenous 7-DHC with methyl-ß-cyclodextrin treatment enhances Hedgehog activation by a pathway agonist. Conversely, exogenous addition of 3ß,5α-dihydroxycholest-7-en-6-one, a naturally occurring B-ring oxysterol derived from 7-DHC that also accumulates in Smith-Lemli-Opitz syndrome, blocked Hedgehog signaling by inhibiting activation of the essential transduction component Smoothened, through a mechanism distinct from Smoothened modulation by other lipids.

Identification and characterization of prescription drugs that change levels of 7-dehydrocholesterol and desmosterol.

Regulating blood cholesterol (Chol) levels by pharmacotherapy has successfully improved cardiovascular health. There is growing interest in the role of Chol precursors in the treatment of diseases. One sterol precursor, desmosterol (Des), is a potential pharmacological target for inflammatory and neurodegenerative disorders. However, elevating levels of the precursor 7-dehydrocholesterol (7-DHC) by inhibiting the enzyme 7-dehydrocholesterol reductase is linked to teratogenic outcomes. Thus, altering the sterol profile may either increase risk toward an adverse outcome or confer therapeutic benefit depending on the metabolite affected by the pharmacophore. In order to characterize any unknown activity of drugs on Chol biosynthesis, a chemical library of Food and Drug Administration-approved drugs was screened for the potential to modulate 7-DHC or Des levels in a neural cell line. Over 20% of the collection was shown to impact Chol biosynthesis, including 75 compounds that alter 7-DHC levels and 49 that modulate Des levels. Evidence is provided that three tyrosine kinase inhibitors, imatinib, ponatinib, and masitinib, elevate Des levels as well as other substrates of 24-dehydrocholesterol reductase, the enzyme responsible for converting Des to Chol. Additionally, the mechanism of action for ponatinib and masitinib was explored, demonstrating that protein levels are decreased as a result of treatment with these drugs.