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Cardiovascular disease risk depends on high-density lipoprotein (HDL) function, not HDL-cholesterol. Isolevuglandins (IsoLGs) are lipid dicarbonyls that react with lysine residues of proteins and phosphatidylethanolamine. IsoLG adducts are elevated in atherosclerosis. The consequences of IsoLG modification of HDL have not been studied. We hypothesized that IsoLG modification of apoA-I deleteriously alters HDL function. We determined the effect of IsoLG on HDL structure-function and whether pentylpyridoxamine (PPM), a dicarbonyl scavenger, can preserve HDL function. IsoLG adducts in HDL derived from patients with familial hypercholesterolemia ( = 10, 233.4 ± 158.3 ng/mg) were found to be significantly higher than in healthy controls ( = 7, 90.1 ± 33.4 pg/mg protein). Further, HDL exposed to myeloperoxidase had elevated IsoLG-lysine adducts (5.7 ng/mg protein) compared with unexposed HDL (0.5 ng/mg protein). Preincubation with PPM reduced IsoLG-lysine adducts by 67%, whereas its inactive analogue pentylpyridoxine did not. The addition of IsoLG produced apoA-I and apoA-II cross-links beginning at 0.3 molar eq of IsoLG/mol of apoA-I (0.3 eq), whereas succinylaldehyde and 4-hydroxynonenal required 10 and 30 eq. IsoLG increased HDL size, generating a subpopulation of 16-23 nm. 1 eq of IsoLG decreased HDL-mediated [H]cholesterol efflux from macrophages via ABCA1, which corresponded to a decrease in HDL-apoA-I exchange from 47.4% to only 24.8%. This suggests that IsoLG inhibits apoA-I from disassociating from HDL to interact with ABCA1. The addition of 0.3 eq of IsoLG ablated HDL's ability to inhibit LPS-stimulated cytokine expression by macrophages and increased IL-1β expression by 3.5-fold. The structural-functional effects were partially rescued with PPM scavenging.
© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
We applied isotopically nonstationary C metabolic flux analysis (INST-MFA) to compare the pathway fluxes of wild-type (WT) Synechococcus elongatus PCC 7942 to an engineered strain (SA590) that produces isobutyraldehyde (IBA). The flux maps revealed a potential bottleneck at the pyruvate kinase (PK) reaction step that was associated with diversion of flux into a three-step PK bypass pathway involving the enzymes PEP carboxylase (PEPC), malate dehydrogenase (MDH), and malic enzyme (ME). Overexpression of pk in SA590 led to a significant improvement in IBA specific productivity. Single-gene overexpression of the three enzymes in the proposed PK bypass pathway also led to improvements in IBA production, although to a lesser extent than pk overexpression. Combinatorial overexpression of two of the three genes in the proposed PK bypass pathway (mdh and me) led to improvements in specific productivity that were similar to those achieved by single-gene pk overexpression. Our work demonstrates how C flux analysis can be used to identify potential metabolic bottlenecks and novel metabolic routes, and how these findings can guide rational metabolic engineering of cyanobacteria for increased production of desired molecules.
Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.
Rapidly proliferating cells require an increased rate of metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway, and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is strongly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly termed the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 in vitro, including Cys424 and His439, which play a role in protein-protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of the electrophile, most notably in the case of ONE, result in substantial protein-protein cross-linking in vitro and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation.
AIMS - Products of lipid oxidation, such as 4-hydroxynonenal (4-HNE), are key activators of hepatic stellate cells (HSC) to a pro-fibrogenic phenotype. Isolevuglandins (IsoLG) are a family of acyclic γ-ketoaldehydes formed through oxidation of arachidonic acid or as by-products of the cyclooxygenase pathway. IsoLGs are highly reactive aldehydes which are efficient at forming protein adducts and cross-links at concentrations 100-fold lower than 4-hydroxynonenal. Since the contribution of IsoLGs to liver injury has not been studied, we synthesized 15-E-IsoLG and used it to investigate whether IsoLG could induce activation of HSC.
RESULTS - Primary human HSC were exposed to 15-E-IsoLG for up to 48h. Exposure to 5μM 15-E-IsoLG in HSCs promoted cytotoxicity and apoptosis. At non-cytotoxic doses (50 pM-500nM) 15-E-IsoLG promoted HSC activation, indicated by increased expression of α-SMA, sustained activation of ERK and JNK signaling pathways, and increased mRNA and/or protein expression of cytokines and chemokines, which was blocked by inhibitors of JNK and NF-kB. In addition, IsoLG promoted formation of reactive oxygen species, and induced an early activation of ER stress, followed by autophagy. Inhibition of autophagy partially reduced the pro-inflammatory effects of IsoLG, suggesting that it might serve as a cytoprotective response.
INNOVATION - This study is the first to describe the biological effects of IsoLG in primary HSC, the main drivers of hepatic fibrosis.
CONCLUSIONS - IsoLGs represent a newly identified class of activators of HSC in vitro, which are biologically active at concentrations as low as 500 pM, and are particularly effective at promoting a pro-inflammatory response and autophagy.
Copyright © 2016. Published by Elsevier Inc.
Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles. ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 protein-protein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14-3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0.1:1 or less. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14-3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Oxidative stress is a contributing factor in a number of chronic diseases, including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation that occurs during periods of oxidative stress results in the formation of lipid electrophiles, which can modify a multitude of proteins in the cell. 4-Hydroxy-2-nonenal (HNE) is one of the most well-studied lipid electrophiles and has previously been shown to arrest cells at the G1/S transition. Recently, proteomic data have shown that HNE is capable of covalently modifying CDK2, the kinase responsible for the G1/S transition. Here, we identify the sites adducted by HNE using recombinant CDK2 and show that HNE treatment suppresses the kinase activity of the enzyme. We further identify sites of adduction in HNE-treated intact human colorectal carcinoma cells (RKO) and show that HNE-dependent modification in cells is long-lived, disrupts CDK2 function, and correlates with a delay of progression of the cells into S-phase. We propose that adduction of CDK2 by HNE directly alters its activity, contributing to the cell cycle delay.
Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids-salicyluric acid and gentisuric acid-and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1.
Copyright © 2015 Elsevier B.V. All rights reserved.
AIMS - Increased lipid peroxidation occurs in many conditions associated with inflammation. Because lipid peroxidation produces lipid aldehydes that can induce inflammatory responses through unknown mechanisms, elucidating these mechanisms may lead to development of better treatments for inflammatory diseases. We recently demonstrated that exposure of cultured cells to lipid aldehydes such as isolevuglandins (IsoLG) results in the modification of phosphatidylethanolamine (PE). We therefore sought to determine (i) whether PE modification by isolevuglandins (IsoLG-PE) occurred in vivo, (ii) whether IsoLG-PE stimulated the inflammatory responses of macrophages, and (iii) the identity of receptors mediating the inflammatory effects of IsoLG-PE.
RESULTS - IsoLG-PE levels were elevated in plasma of patients with familial hypercholesterolemia and in the livers of mice fed a high-fat diet to induce obesity and hepatosteatosis. IsoLG-PE potently stimulated nuclear factor kappa B (NFκB) activation and expression of inflammatory cytokines in macrophages. The effects of IsoLG-PE were blocked by the soluble form of the receptor for advanced glycation endproducts (sRAGE) and by RAGE antagonists. Furthermore, macrophages derived from the bone marrow of Ager null mice failed to express inflammatory cytokines in response to IsoLG-PE to the same extent as macrophages from wild-type mice.
INNOVATION - These studies are the first to identify IsoLG-PE as a mediator of macrophage activation and a specific receptor, RAGE, which mediates its biological effects.
CONCLUSION - PE modification by IsoLG forms RAGE ligands that activate macrophages, so that the increased IsoLG-PE generated by high circulating cholesterol levels or high-fat diet may play a role in the inflammation associated with these conditions.
Products of oxidative damage to lipids include 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE), both of which are cytotoxic electrophiles. ONE reacts more rapidly with nucleophilic amino acid side chains, resulting in covalent protein adducts, including residue-residue cross-links. Previously, we demonstrated that peptidylprolyl cis/trans isomerase A1 (Pin1) was highly susceptible to adduction by HNE and that the catalytic cysteine (Cys113) was the preferential site of modification. Here, we show that ONE also preferentially adducts Pin1 at the catalytic Cys but results in a profoundly different modification. Results from experiments using purified Pin1 incubated with ONE revealed the principal product to be a Cys-Lys pyrrole-containing cross-link between the side chains of Cys113 and Lys117. In vitro competition assays between HNE and ONE demonstrate that ONE reacts more rapidly than HNE with Cys113. Exposure of RKO cells to alkynyl-ONE (aONE) followed by copper-mediated click chemistry and streptavidin purification revealed that Pin1 is also modified by ONE in cells. Analysis of the Pin1 crystal structure reveals that Cys113 and Lys117 are oriented toward each other in the active site, facilitating formation of an ONE cross-link.
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.