Polyunsaturated fatty acids (PUFA) are primary targets of free radical damage during oxidative stress. Diffusible electrophilic R, -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 streptavidincoated 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 glucoseregulated 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.

Biological electrophiles result from oxidative metabolism of exogenous compounds or endogenous cellular constituents, and they contribute to pathophysiologies such as toxicity and carcinogenicity. The chemical toxicology of electrophiles is dominated by covalent addition to intracellular nucleophiles. Reaction with DNA leads to the production of adducts that block replication or induce mutations. The chemistry and biology of electrophile−DNA reactions have been extensively studied, providing in many cases a detailed understanding of the relation between adduct structure and mutational consequences. By contrast, the linkage between protein modification and cellular response is poorly understood.In this Account, we describe our efforts to define the chemistry of protein modification and its biological consequences using lipid-derived α,β-unsaturated aldehydes as model electrophiles. In our global approach, two large data sets are analyzed: one represents the identity of proteins modified over a wide range of electrophile concentrations, and the second comprises changes in gene expression observed under similar conditions. Informatics tools show theoretical connections based primarily on transcription factors hypothetically shared between the two data sets, downstream of adducted proteins and upstream of affected genes. This method highlights potential electrophile-sensitive signaling pathways and transcriptional processes for further evaluation.Peroxidation of cellular phospholipids generates a complex mixture of both membrane-bound and diffusible electrophiles. The latter include reactive species such as malondialdehyde, 4-oxononenal, and 4-hydroxynonenal (HNE). Enriching HNE-adducted proteins for proteomic analysis was a technical challenge, solved with click chemistry that generated biotin-tagged protein adducts. For this purpose, HNE analogues bearing terminal azide or alkyne functionalities were synthesized. Cellular lysates were first exposed to a single type of HNE analogue (azido- or alkynyl-HNE), and then click reactions were performed against the cognate alkynyl- and azido-biotin derivative. The resulting biotin-labeled proteins were captured and enriched over a streptavidin matrix for subsequent mass spectrometric analysis. We thereby identified a multitude of HNE targets. Simultaneous microarray analysis of changes in gene expression triggered by HNE also produced an abundance of data. Functional analysis of both data sets generated the hypothesis that an important pathway of cellular response derives from electrophile modification of protein chaperones, resulting in the release of transcription factors that are their clients. Informatic analysis of the protein modification and microarray data sets identified several transcription factors as potential mediators of the cellular response to HNE-adducted proteins. Among these, heat shock factor 1 (HSF1) was confirmed as a sensitive and robust effector of HNE-induced changes in gene expression. Activation of HSF1 appears, in part, to be mediated by the el...

The production of nitric oxide by macrophages has been implicated as a host defense mechanism against microbial pathogens and tumor cells. Recent reports have implicated interferon-␣/␤ (IFN-␣/␤) as an autocrine/paracrine signal critical for the induction of murine iNOS. In this report we have systematically investigated the role of IFN-␤ in the induction of iNOS in the murine macrophage cell line, RAW 264.7. First, we demonstrate that IFN-␤ expression is highly up-regulated, and is secreted in response to lipopolysaccharide (LPS). Treatment of RAW macrophages with LPS results in a time-dependent phosphorylation of STAT-1 on both tyrosine residue 701 (Tyr-701) and serine residue 727 (Ser-727) that is consistent with the timing of endogenous IFN-␤ expression. LPS also induces interferon regulatory factor-1 expression with similar kinetics. We further demonstrate that exogenous IFN-␤ accelerates the induction of iNOS by LPS. The acceleration of iNOS induction is observed at the levels of transcription, protein expression, and NO formation. Accordingly, we propose that the cytokine environment of macrophages may determine the rate and magnitude of nitric oxide production, thereby regulating the cytotoxic response to pathogen challenge. Activated macrophages produce nitric oxide (NO), 1 a free radical species that mediates cytotoxic and cytostatic effects against pathogenic microbes and tumor cells (1-3). The enzyme responsible for the production of NO by macrophages is the inducible isoform of nitric-oxide synthase (iNOS), which catalyzes the oxidation of one of the equivalent guanidino nitrogens of arginine to form NO and citrulline (4). Compared with the neuronal and endothelial isoforms of NOS (nNOS and eNOS, respectively), iNOS generates high concentrations of NO, which accounts for the cytotoxic and cytostatic effects of NO on target cells (5). The regulation of NO production by iNOS has been extensively studied in murine macrophages, and occurs primarily at the level of transcription. The gene encoding iNOS is transcriptionally silent, but is dramatically induced by bacterial lipopolysaccharide (LPS). LPS activates Toll-like receptordependent signaling pathways in macrophages, including the transcription factor, NF-B (6). However, maximal induction is not achieved by LPS alone. The induction of iNOS by LPS can be synergized by the addition of specific cytokines, including tumor necrosis factor-␣, interleukin-1␤, and interferon-␥ (IFN-␥) (7). This synergy is reflected in the promoter region of the murine iNOS gene, which contains oligonucleotide motifs for the binding of both LPS-and several cytokine-induced transcription factors (8).Both type I (IFN-␣/␤) and type II (IFN-␥) interferons mediate the induction of target genes via the activation of the JAK-STAT signaling pathway (9). Signal transducer and activator of transcription (STAT) proteins are a family of transcriptional activators that interact with IFN receptors and associated Janus kinase (JAK) proteins. The activation of STAT proteins is regulated by p...

The objectives of this study were to determine whether rat aortic smooth muscle cells (RASMC) express arginase and to elucidate the possible mechanisms involved in the regulation of arginase expression. The results show that RASMC contain basal arginase I (AI) activity, which is significantly enhanced by stimulating the cells with either interleukin (IL)-4 or IL-13, but arginase II (AII) expression was not detected under any condition studied here. We further investigated the signal transduction pathways responsible for AI induction. AI mRNA and protein levels were enhanced by addition of forskolin (1 μM) and inhibited by H-89 (30 μM), suggesting positive regulation of AI by a protein kinase A pathway. Genistein (10 μg/ml) and sodium orthovanadate (Na3VO4; 10 μM) were used to investigate the role of tyrosine phosphorylation in the control of AI expression. Genistein inhibited, whereas Na3VO4enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon (IFN)-γ were investigated for their effects on AI induction. Dex (1 μM) and IFN-γ (100 U/ml) alone had no effect on basal AI expression in RASMC, but both reduced AI induction by IL-4 and IL-13. In combination, Dex and IFN-γ abolished AI induction by IL-4 and IL-13. Finally, both IL-4 and IL-13 significantly increased RASMC DNA synthesis as monitored by [3H]thymidine incorporation, demonstrating that upregulation of AI is correlated with an increase in cell proliferation. Blockade of AI induction by IFN-γ, H-89, or genistein also blocked the increase in cell proliferation. These observations are consistent with the possibility that upregulation of AI might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

4-Hydroxynonenal (HNE) is a pro-apoptotic electrophile generated during the spontaneous decomposition of oxidized lipids. We have previously shown that HNE activates the transcription factor, heat shock factor 1 (HSF1), and promotes cytoprotective heat shock gene expression and that silencing HSF1 sensitizes the colon cancer cell line RKO to HNE-induced apoptosis. Here we report a reduction in the anti-apoptotic proteins Bcl-X L , Mcl-1, and Bcl-2 in HSF1-silenced RKO cells, and we examine the underlying mechanism. To investigate the regulation of the Bcl-2 family by HSF1, microarray analysis of gene expression was performed. We observed that the Hsp70 co-chaperone, BAG3 (Bcl-2-associated athanogene domain 3), is strongly induced by HNE in control but not in HSF1-silenced colon cancer cells. Silencing BAG3 expression with small interfering RNA caused a dramatic reduction in Bcl-X L , Mcl-1, and Bcl-2 protein levels in colon cancer cells and increased apoptosis, similar to the effect of silencing HSF1. Also, immunoprecipitation experiments indicate specific interactions between BAG3, Hsp70, and the Bcl-2 family member, Bcl-X L . Overall, our data reveal that BAG3 is HSF1-inducible and has a unique role facilitating cancer cell survival during pro-apoptotic stress by stabilizing the level of Bcl-2 family proteins.The indiscriminate oxidation of biological membranes is increasingly recognized as a contributing factor in a variety of neurodegenerative and inflammatory diseases, including Alzheimer and Parkinson diseases, atherosclerosis, diabetes, alcoholic liver disease, and cancer (1-5). An established route for lipid oxidation involves hydrogen atom abstraction from unsaturated fatty acids by reactive oxidants, followed by reaction with oxygen to form lipid hydroperoxides (6). Various electrophiles, including the ␣,␤-unsaturated aldehyde 4-hydroxynonenal (HNE), 2 are generated from the nonenzymatic decomposition of lipid hydroperoxides (7). HNE is a diffusible lipid species and is capable of reacting with nucleophiles throughout the cell, such as glutathione, nucleic acids, and proteins (8).HNE promotes apoptosis in mammalian cells at low to mid-micromolar concentrations (9 -11). In response to HNE, cells also activate cytoprotective pathways that abrogate programmed cell death. Microarray analysis of gene expression highlights the variety of protective measures activated in response to HNE, including DNA damage, antioxidant, heat shock, and ER stress pathways (12). The heat shock response, mediated by the transcription factor HSF1, is strongly activated in HNE-treated cells. We previously reported that HNE elicits the nuclear translocation of HSF1 and promotes Hsp40 (DNAJB1) and Hsp72 (HspA1A) expression (13). Using siRNA to silence HSF1, we found that cells deficient in heat shock gene expression are dramatically sensitized to the pro-apoptotic effects of HNE. Investigating the mechanism of cell death in HSF1-silenced cells, we discovered that Bcl-X L protein levels are significantly reduced, thereby predisposi...