Keri A. Tallman

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 alpha,beta-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 microM. 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.

Free Radical Oxidation of Polyunsaturated Lipids: New Mechanistic Insights and the Development of Peroxyl Radical Clocks

The peroxidation of lipids in biological membranes has been implicated in both the onset and development of most degenerative diseases. The primary products of this autoxidation process are usually lipid hydroperoxides. They form as a consequence of a free radical chain reaction: lipid peroxyl radicals propagate the chain by rate-limiting H-atom abstraction from another lipid. Studies of the mechanism of lipid peroxidation are a specific part of a wider effort to understand the more general phenomenon of hydrocarbon autoxidation, which dates back some 70 years. However, the autoxidation of lipids is generally much more complicated than that of other hydrocarbons because of additional reaction pathways afforded by a variety of uniquely positioned unsaturated bonds. Indeed, polyunsaturation is an important aspect of many of the most relevant of physiological lipids, such as linoleate and arachidonate. In this Account, we present our current understanding of the mechanism of unsaturated lipid peroxidation, effectively updating our Account on the same topic published 25 years ago. Our more recent work has, in large part, been stimulated by the discovery of the nonconjugated linoleate hydroperoxide as a product under certain autoxidation conditions. The identification of this long-elusive bis-allylic hydroperoxide prompted our kinetic characterization of the reaction leading to its formation. The product distributions obtained from autoxidations of newly synthesized model compounds, which vary in either the substitution of the bis-allylic moiety or the configuration of the double bonds, have provided key insights into the overall mechanism. These insights have in turn been reinforced by the results of theoretical calculations. The picture that emerges is one wherein the delocalized carbon-centered radicals, which arise as intermediates in these reactions, first associate with dioxygen to form pre-reaction complexes. These complexes then collapse through transition state structures that maximize the orbital interactions between the delocalized radical SOMO and dioxygen. The energies of these transition states are influenced by steric effects; thus, there are distinct changes in product distribution in the autoxidation of dienes having different substitution patterns. The radical-dioxygen complexes are also intermediates in the isomerization of allylperoxyl and pentadienylperoxyls, helping explain the high regio- and stereochemical fidelity of these processes. We have taken advantage of the rapid fragmentation of nonconjugated peroxyl radicals to develop a powerful peroxyl radical clock methodology, which can be used to determine rate constants for reactions of peroxyl radicals with molecules having rate constants ranging from 1 to 10(7) M(-1) s(-1). We can make use of this methodology to address various questions, both fundamental and applied, relating to lipid peroxidation and its inhibition by radical-trapping antioxidants.

An Azido-Biotin Reagent for Use in the Isolation of Protein Adducts of Lipid-derived Electrophiles by Streptavidin Catch and Photorelease

HNE (4-hydroxynonenal), a byproduct of lipid peroxidation, reacts with nucleophilic centers on proteins. A terminal alkynyl analog of HNE (alkynyl HNE, aHNE) serves as a surrogate for HNE itself, both compounds reacting with protein amine and thiol functional groups by similar chemistry. Proteins modified with aHNE undergo reaction with a click reagent that bears azido and biotin groups separated by a photocleavable linker. Peptides and proteins modified in this way are affinity purified on streptavidin beads. Photolysis of the beads with a low intensity UV light releases bound biotinylated proteins or peptides, i.e. proteins or peptides modified by aHNE. Two strategies, (a) protein catch and photorelease and (b) peptide catch and photorelease, are employed to enrich adducted proteins or peptide mixtures highly enriched in adducts. Proteomics analysis of the streptavidin-purified peptides by LC-MS/MS permits identification of the adduction site. Identification of 30 separate peptides from human serum albumin by peptide catch and photorelease reveals 18 different aHNE adduction sites on the protein. Protein catch and photorelease shows that both HSA and ApoA1 in human plasma undergo significant modification by aHNE.

Global, in situ, site-specific analysis of protein S-sulfenylation

Protein S-sulfenylation is the reversible oxidative modification of cysteine thiol groups to form cysteine S-sulfenic acids. Mapping the specific sites of protein S-sulfenylation onto complex proteomes is crucial to understanding the molecular mechanisms controlling redox signaling and regulation. This protocol describes global, in situ, site-specific analysis of protein S-sulfenylation using sulfenic acid–specific chemical probes and mass spectrometry (MS)-based proteomics. The major steps in this protocol are as follows: (i) optimization of conditions for selective labeling of cysteine S-sulfenic acids in intact cells with the commercially available dimedone-based probe, DYn-2; (ii) tagging the modified cysteines with a functionalized biotin reagent containing a cleavable linker via Cu(I)-catalyzed azide-alkyne cycloaddition reaction; (iii) enrichment of the biotin-tagged tryptic peptides with streptavidin; (iv) liquid chromatography-tandem MS (LC-MS/MS)-based shotgun proteomics; and (v) computational data analysis. We also outline strategies for quantitative analysis of this modification in cells responding to redox perturbations and discuss special issues pertaining to experimental design of thiol redox studies. Our chemoproteomic platform should be broadly applicable to the investigation of other bio-orthogonal chemically engineered post-translational modifications. The entire analysis protocol takes ∼1 week to complete.

Alkylation damage by lipid electrophiles targets functional protein systems

Protein alkylation by reactive electrophiles contributes to chemical toxicities and oxidative stress, but the functional impact of alkylation damage across proteomes is poorly understood. We used Click chemistry and shotgun proteomics to profile the accumulation of proteome damage in human cells treated with lipid electrophile probes. Protein target profiles revealed three damage susceptibility classes, as well as proteins that were highly resistant to alkylation. Damage occurred selectively across functional protein interaction networks, with the most highly alkylation-susceptible proteins mapping to networks involved in cytoskeletal regulation. Proteins with lower damage susceptibility mapped to networks involved in protein synthesis and turnover and were alkylated only at electrophile concentrations that caused significant toxicity. Hierarchical susceptibility of proteome systems to alkylation may allow cells to survive sublethal damage while protecting critical cell functions.

Conversion of 7-Dehydrocholesterol to 7-Ketocholesterol Is Catalyzed by Human Cytochrome P450 7A1 and Occurs by Direct Oxidation without an Epoxide Intermediate

7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (kcat/Km = 3 × 104 m−1 s−1). P450 7A1 converted lathosterol (Δ5-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (∼1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a FeIII-O-C-C+ intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482–5489). Accordingly, reaction of P450 7A1 with 7-[2H1]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.

Stable histone adduction by 4-oxo-2-nonenal: a potential link between oxidative stress and epigenetics.

Lipid electrophiles modify cellular targets, altering their function. Here, we describe histones as major targets for modification by 4-oxo-2-nonenal, resulting in a stable Lys modification structurally analogous to other histone Lys acylations. Seven adducts were identified in chromatin isolated from intact cells: four 4-ketoamides to Lys and three Michael adducts to His. A 4-ketoamide adduct residing at H3K27 was identified in stimulated macrophages. Modification of histones H3 and H4 prevented nucleosome assembly.

Capture and release of alkyne-derivatized glycerophospholipids using cobalt chemistry

Alkyne modified phospholipids can be unambiguously identified and differentiated from native species in complex mixtures by formation of dicobalthexacarbonyl complexes. This reaction is specific for alkynes and is unaffected by other glycerophospholipid related moieties. Enrichment of cells with alkyne-derivatized fatty acids or glycerophospholipids followed by solid phase sequestration and release is a promising new method for unequivocally monitoring individual glycerophospholipids following incorporation and facilitates lipidomic analysis of substrates and products.

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 in situ. 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.

Quantitative assays for esterified oxylipins generated by immune cells

Phospholipid-esterified oxylipins include newly described families of bioactive lipids generated by lipoxygenases in immune cells. Until now, assays for their quantitation were not well developed or widely available. Here, we describe a mass spectrometric protocol that enables accurate measurement of several esterified oxylipins—in particular hydro(pero)xyeicosatetraenoic acids, hydroxyoctadecadienoic acids, hydroxydocosahexaenoic acids and keto-eicosatetraenoic acids—attached to either phosphatidylethanolamine or phosphatidylcholine. Lipids are isolated from cells or tissue using a liquid-phase organic extraction, then analyzed by HPLC–tandem mass spectrometry (LC/MS/MS) in multiple reaction–monitoring mode. The protocol can simultaneously monitor up to 23 species. Generation of standards takes ∼2 d. Following this, extraction of 30 samples takes ∼3 h, with LC/MS/MS run time of 50 min per sample.