Reactivities and Products of Free Radical Oxidation of Cholestadienols

7-Dehydrocholesterol (7-DHC) is the most oxidizable lipid molecule reported to date, with a propagation rate constant for free radical peroxidation that is 200 times that of cholesterol. To better understand the high reactivity of 7-DHC and elucidate the reaction mechanism, we synthesized conjugated and skipped nonconjugated cholestadienols that would give one of the two putative pentadienyl-radical intermediates formed in 7-DHC peroxidation. The additional dienols include 6,8(9)-dienol, 5,8(14)-dienol, 6,8(14)-dienol, and the biologically important 8-dehydro­chole­sterol (8-DHC; 5,8(9)-dienol). We found that all of the dienols are significantly (at least 40 times) more reactive than cholesterol. Among them, dienols leading to the formation of the pentadienyl radical in ring B (termed <i><b>endo-B</b></i>) of the sterol are more reactive than those leading to the pentadienyl radical spanning rings B and C (termed <i><b>exo-B</b></i>). By comparing the oxysterol profile formed from 7-DHC and those formed from 8-DHC and 5,8(14)-dienol, products formed from abstraction of the hydrogen atoms at C-9 and C-14 (<i>H-9</i> or <i>H-14 mechanism</i>) were clearly differentiated. When the oxidation was carried out in the presence of the good hydrogen atom donor α-tocopherol, the oxysterol profile of 7-DHC peroxidation differed distinctly from the profile observed in the absence of the antioxidant and resembles more closely the profile observed in biological systems. This study suggests that oxidative stress and the accumulation of oxysterols should be considered as two key factors in cholesterol biosynthesis or metabolism disorders, where dienyl sterol intermediates are accumulated

Quantitative Chemoproteomics for Site-Specific Analysis of Protein Alkylation by 4‑Hydroxy-2-Nonenal in Cells

Protein alkylation by 4-hydroxy-2-nonenal (HNE), an endogenous lipid derived electrophile, contributes to stress signaling and cellular toxicity. Although previous work has identified protein targets for HNE alkylation, the sequence specificity of alkylation and dynamics in a cellular context remain largely unexplored. We developed a new quantitative chemoproteomic platform, which uses isotopically tagged, photocleavable azido-biotin reagents to selectively capture and quantify the cellular targets labeled by the alkynyl analogue of HNE (aHNE). Our analyses site-specifically identified and quantified 398 aHNE protein alkylation events (386 cysteine sites and 12 histidine sites) in intact cells. This data set expands by at least an order of magnitude the number of such modification sites previously reported. Although adducts formed by Michael addition are thought to be largely irreversible, we found that most aHNE modifications are lost rapidly <i>in situ</i>. Moreover, aHNE adduct turnover occurs only in intact cells and loss rates are site-selective. This quantitative chemoproteomics platform provides a versatile general approach to map bioorthogonal-chemically engineered post-translational modifications and their cellular dynamics in a site-specific and unbiased manner

Structure−Activity Analysis of Diffusible Lipid Electrophiles Associated with Phospholipid Peroxidation: 4-Hydroxynonenal and 4-Oxononenal Analogues

Electrophile-mediated disruption of cell signal-ing is involved in the pathogenesis of several diseases including atherosclerosis and cancer. Diffusible and membrane bound lipid electrophiles are known to modify DNA and protein substrates and modulate cellular pathways including ER stress, antioxidant response, DNA damage, heat shock, and apoptosis. Herein we report on a structure−activity relationship for several electrophilic analogues of 4-hydroxynonenal (HNE) and 4-oxononenal (ONE) with regard to toxicity and anti-inflammatory activity. The analogues studied were the oxidation products of HNE and ONE, HNEA/ONEA, the <i>in vivo</i> hydrolysis products of oxidized phosphatidylcholine, COOH-HNE/COOH-ONE, and their methyl esters, COOMe-HNE/ONE. The reactivity of each compound toward <i>N</i>-acetylcysteine was determined and compared to the toxicity toward a human colorectal carcinoma cell line (RKO) and a human monocytic leukemia cell line (THP-1). Further analysis was performed in differentiated THP-1 macrophages to assess changes in macrophage activation and pro-inflammatory signaling in response to each lipid electrophile. HNE/ONE analogues inhibited THP-1 macrophage production of the pro-inflammatory cytokines, IL-6, IL-1β, and TNFα, after lipopolysaccharide (LPS)/IFNγ activation. Inhibition of cytokine production was observed at submicromolar concentrations of several analogues with as little as 30 min of exposure. Phagocytosis of fluorescent beads was also inhibited by lipid electrophile treatment. Lipid electrophiles related to HNE/ONE are both toxic and anti-inflammatory, but the anti-inflammatory effects in human macrophages are observed at nontoxic concentrations. Neither toxicity nor anti-inflammatory activity are strongly correlated to the reactivity of the model nucleophile, <i>N</i>-acetylcysteine

Identification of Proteins Adducted by Lipid Peroxidation Products in Plasma and Modifications of Apolipoprotein A1 with a Novel Biotinylated Phospholipid Probe

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidycholine probe called PLPBSO (Tallman et al. Chem. Res. Toxicol. 2007, 20, 227−234), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS−MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS−MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10(<i>E</i>)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10(<i>E</i>)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress

Activity-Based Probes Linked with Laser-Cleavable Mass Tags for Signal Amplification in Imaging Mass Spectrometry: Analysis of Serine Hydrolase Enzymes in Mammalian Tissue

A novel functional imaging mass spectrometry technology is described that utilizes activity-based probes for imaging enzyme active sites in tissue sections. We demonstrate this technology using an activity-based probe (fluorophosphate) that is specific for serine hydrolases. A dendrimer containing multiple mass tags that is attached to the activity-based probe is used to analyze the binding sites of the probe through release and measurement of the mass tags on laser irradiation. A generation 8 poly­(amido amine) dendrimer with 1024 amino groups was labeled with an azide group, and then, more than 900 mass tags were attached in order to achieve signal amplification of nearly 3 orders of magnitude. The experimental protocol first involves binding of the activity-based probe containing an alkyne group to serine hydrolases in the tissue section followed by attachment of the dendrimer labeled with mass tags to the bound probe by Click chemistry. On irradiation of the labeled tissue by the laser beam in a raster pattern, the mass tags are liberated and recorded by the mass analyzer; consequently, the ion image of the mass tag reveals the distribution of serine hydrolases in the tissue. This process was shown using rat brain and mouse embryo sections. Targeted imaging has the advantage of providing high spatial resolution and high sensitivity through the use of signal amplification chemistry with high target specificity through the use of an enzyme activity probe

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

Identification of Protein Targets of 4-Hydroxynonenal Using Click Chemistry for ex Vivo Biotinylation of Azido and Alkynyl Derivatives

Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic α,β-unsaturated aldehydes, such as 4-hydroxynonenal (HNE), have been shown to modify proteins that mediate cell signaling (e.g., IKK and Keap1) and alter gene expression pathways responsible for inducing antioxidant genes, heat shock proteins, and the DNA damage response. To fully understand cellular responses to HNE, it is important to determine its protein targets in an unbiased fashion. This requires a strategy for detecting and isolating HNE-modified proteins regardless of the nature of the chemical linkage between HNE and its targets. Azido or alkynyl derivatives of HNE were synthesized and demonstrated to be equivalent to HNE in their ability to induce heme oxygenase induction and induce apoptosis in colon cancer (RKO) cells. Cells exposed to the tagged HNE derivatives were lysed and exposed to reagents to effect Staudinger ligation or copper-catalyzed Huisgen 1,3 dipolar cycloaddition reaction (click chemistry) to conjugate HNE-adducted proteins with biotin for subsequent affinity purification. Both strategies yielded efficient biotinylation of tagged HNE−protein conjugates, but click chemistry was found to be superior for the recovery of biotinylated proteins from streptavidin-coated beads. Biotinylated proteins were detected in lysates from RKO cell incubations with azido-HNE at concentrations as low as 1 µM. These proteins were affinity purified with streptavidin beads, and proteomic analysis was performed by linear ion trap mass spectrometry. Proteomic analysis revealed a dose-dependent increase in labeled proteins with increased sequence coverage at higher concentrations. Several proteins involved in stress signaling (heat shock proteins 70 and 90 and the 78-kDa glucose-regulated protein) were selectively adducted by azido- and alkynyl-HNE. The use of azido and alkynyl derivatives in conjunction with click chemistry appears to be a valuable approach for the identification of the protein targets of HNE

Inhibitors of 7‑Dehydrocholesterol Reductase: Screening of a Collection of Pharmacologically Active Compounds in Neuro2a Cells

A small library of pharmacologically active compounds (the NIH Clinical Collection) was assayed in Neuro2a cells to determine their effect on the last step in the biosynthesis of cholesterol, the transformation of 7-dehydrocholesterol (7-DHC) to cholesterol promoted by 7-dehydrocholesterol reductase, <i>DHCR7.</i> Of some 727 compounds in the NIH Clinical Collection, over 30 compounds significantly increased 7-DHC in Neuro2a cells when assayed at 1 μM. Active compounds that increased 7-DHC with a <i>Z-</i>score of +3 or greater generally gave rise to modest decreases in desmosterol and increases in lanosterol levels. Among the most active compounds identified in the library were the antipsychotic, antidepressant, and anxiolytic compounds that included perospirone, nefazodone, haloperidol, aripiprazole, trazodone, and buspirone. Fluoxetine and risperidone were also active at 1 μM, and another 10 compounds in this class of pharmaceuticals were identified in the screen at concentrations of 10 μM. Increased levels of 7-DHC are associated with Smith-Lemli-Opitz syndrome (SLOS), a human condition that results from a mutation in the gene that encodes <i>DHCR7.</i> The SLOS phenotype includes neurological deficits and congenital malformations, and it is linked to a higher incidence of autism spectrum disorder. The significance of the current study is that it identifies common pharmacological compounds that may induce a biochemical presentation similar to SLOS. Little is known about the side effects of elevated 7-DHC postdevelopmentally, and the elevated 7-DHC that results from exposure to these compounds may also be a confounder in the diagnosis of SLOS

Assay of Protein and Peptide Adducts of Cholesterol Ozonolysis Products by Hydrophobic and Click Enrichment Methods

Cholesterol undergoes ozonolysis to afford a variety of oxysterol products, including cholesterol-5,6-epoxide (CholEp) and the isomeric aldehydes secosterol A (seco A) and secosterol B (seco B). These oxysterols display numerous important biological activities, including protein adduction; however, much remains to be learned about the identity of the reactive species and the range of proteins modified by these oxysterols. Here, we synthesized alkynyl derivatives of cholesterol-derived oxysterols and employed a straightforward detection method to establish secosterols A and B as the most protein-reactive of the oxysterols tested. Model adduction studies with an amino acid, peptides, and proteins provide evidence for the potential role of secosterol dehydration products in protein adduction. Hydrophobic separation methodsFolch extraction and solid phase extraction (SPE)were successfully applied to enrich oxysterol-adducted peptide species, and LC-MS/MS analysis of a model peptide–seco adduct revealed a unique fragmentation pattern (neutral loss of 390 Da) for that species. Coupling a hydrophobic enrichment method with proteomic analysis utilizing characteristic fragmentation patterns facilitates the identification of secosterol-modified peptides and proteins in an adducted protein. More broadly, these improved enrichment methods may give insight into the role of oxysterols and ozone exposure in the pathogenesis of a variety of diseases, including atherosclerosis, Alzheimer’s disease, Parkinson’s disease, and asthma

A Chemoproteomic Platform To Assess Bioactivation Potential of Drugs

Reactive metabolites (RM) formed from bioactivation of drugs can covalently modify liver proteins and cause mechanism-based inactivation of major cytochrome P450 (CYP450) enzymes. Risk of bioactivation of a test compound is routinely examined as part of lead optimization efforts in drug discovery. Here we described a chemoproteomic platform to assess <i>in vitro</i> and <i>in vivo</i> bioactivation potential of drugs. This platform enabled us to determine reactivity of thousands of proteomic cysteines toward RMs of diclofenac formed in human liver microsomes and living animals. We pinpointed numerous reactive cysteines as the targets of RMs of diclofenac, including the active (heme-binding) sites on several key CYP450 isoforms (1A2, 2E1 and 3A4 for human, 2C39 and 3A11 for mouse). This general platform should be applied to other drugs, drug candidates, and xenobiotics with potential hepatoxicity, including environmental organic substances, bioactive natural products, and traditional Chinese medicine