Unbiased Identification of Proteins Covalently Modified by Complex Mixtures of Peroxidized Lipids Using a Combination of Electrophoretic Mobility Band Shift with Mass Spectrometry

Covalent modification of functionally important cell proteins by lipid oxidation products (LOPs) is a known mechanism initiating pathological consequences of oxidative stress. Identification of new proteins covalently modified by electrophilic lipids can be performed by a combination of chemical, immunological, and mass spectrometry-based methods, but requires prior knowledge either on the exact molecular structure of LOPs (e.g., 4-hydroxynonenal) or candidate protein targets. However, under the conditions of oxidative stress in vivo, a complex mixture of proteins (e.g., cytosolic proteome) reacts with a complex mixture of LOPs. Here we describe a method for detection of lipid-modified proteins that does not require an a priori knowledge on the chemical structure of LOPs or identity of target proteins. The method is based on the change of electrophoretic mobility of lipid-modified proteins, which is induced by conformational changes and cross-linking with other proteins. Abnormally migrating proteins are detected by mass spectrometry-based protein peptide sequencing. We applied this method to study effects of oxidized palmitoyl-arachidonoyl-phosphatidylcholine (OxPAPC) on endothelial cells. Several known, but also many new, OxPAPC-binding proteins were identified. We expect that this technically relatively simple method can be widely applied for label-free analysis of lipid-protein interactions in complex protein samples treated with different LOPs

Glycosaminoglycan-Mediated Downstream Signaling of CXCL8 Binding to Endothelial Cells

The recruitment of leukocytes, mediated by endothelium bound chemokine gradients, is a vital process in inflammation. The highly negatively charged, unbranched polysaccharide family of glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate mediate chemokine immobilization. Specifically the binding of CXCL8 (interleukin 8) to GAGs on endothelial cell surfaces is known to regulate neutrophil recruitment. Currently, it is not clear if binding of CXCL8 to GAGs leads to endothelial downstream signaling in addition to the typical CXCR1/CXCR2 (C-X-C motif chemokine receptor 1 and 2)-mediated signaling which activates neutrophils. Here we have investigated the changes in protein expression of human microvascular endothelial cells induced by CXCL8. Tumor necrosis factor alpha (TNFα) stimulation was used to mimic an inflammatory state which allowed us to identify syndecan-4 (SDC4) as the potential proteoglycan co-receptor of CXCL8 by gene array, real-time PCR and flow cytometry experiments. Enzymatic GAG depolymerization via heparinase III and chondroitinase ABC was used to emulate the effect of glycocalyx remodeling on CXCL8-induced endothelial downstream signaling. Proteomic analyses showed changes in the expression pattern of a number of endothelial proteins such as Zyxin and Caldesmon involved in cytoskeletal organization, cell adhesion and cell mobility. These results demonstrate for the first time a potential role of GAG-mediated endothelial downstream signaling in addition to the well-known CXCL8-CXCR1/CXCR2 signaling pathways in neutrophils

Langerhans cell maturation is accompanied by induction of N-cadherin and the transcriptional regulators of epithelial-mesenchymal transition ZEB1/2

angerhans cells (LCs) are a unique subset of dendritic cells (DCs) that express epithelial adhesion molecules, allowing them to form contacts with epithelial cells and reside in epidermal/epithelial tissues. The dynamic regulation of epithelial adhesion plays a decisive role in the life cycle of LCs. It controls whether LCs remain immature and sessile within the epidermis or mature and egress to initiate immune responses. So far, the molecular machinery regulating epithelial adhesion molecules during LC maturation remains elusive. Here, we generated pure populations of immature human LCs in vitro to systematically probe for gene-expression changes during LC maturation. LCs down-regulate a set of epithelial genes including E-cadherin, while they upregulate the mesenchymal marker N-cadherin known to facilitate cell migration. In addition, N-cadherin is constitutively expressed by monocyte-derived DCs known to exhibit characteristics of both inflammatory-type and interstitial/dermal DCs. Moreover, the transcription factors ZEB1 and ZEB2 (ZEB is zinc-finger E-box-binding homeobox) are upregulated in migratory LCs. ZEB1 and ZEB2 have been shown to induce epithelial-to-mesenchymal transition (EMT) and invasive behavior in cancer cells undergoing metastasis. Our results provide the first hint that the molecular EMT machinery might facilitate LC mobilization. Moreover, our study suggests that N-cadherin plays a role during DC migration

Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed,Peer reviewe

Low Concentrations of Oxidized Phospholipids Increase Stress Tolerance of Endothelial Cells

Oxidized phospholipids (OxPLs) are generated by enzymatic or autooxidation of esterified polyunsaturated fatty acids (PUFAs) residues. OxPLs are present in circulation and atherosclerotic plaques where they are thought to induce predominantly proinflammatory and toxic changes in endothelial (ECs) and other cell types. Unexpectedly, we found that low concentrations of OxPLs were not toxic but protected ECs from stress induced by serum deprivation or cytostatic drugs. The protective effect was observed in ECs obtained from different vessels and was monitored using a variety of readouts based on different biological and chemical principles. Analysis of the structure–activity relationship identified oxidized or missing fatty acid residue (OxPLs or Lyso-PLs, respectively) as a prerequisite for the protective action of a PL. Protective OxPLs or Lyso-PLs acquired detergent-like properties and formed in solution aggregates 1000 nm, likely multilayer liposomes) produced by nonoxidized precursor PLs. Because surfactants, OxPLs, and Lyso-PLs are known to extract membrane cholesterol, we tested if this effect might trigger the protection of endothelial cells. The protective action of OxPLs and Lyso-PLs was inhibited by cotreatment with cholesterol and mimicked by cholesterol-binding beta-cyclodextrin but not inactive α-cyclodextrin. Wide-scale mRNA expression analysis in four types of ECs showed the induction of genes encoding for heat shock proteins (HSPs) and secreted prosurvival peptides and proteins. Inducers of HSPs, chemical chaperones, and pure prosurvival factors mimicked the protective action of OxPLs/Lyso-PLs. We hypothesize that oxidation changes the physicochemical properties of PLs, thus promoting membrane cholesterol redistribution or extraction leading to the expression of intra- and extracellular prosurvival factors

Oxidized phospholipids on alkyl-amide scaffold demonstrate anti-endotoxin and endothelial barrier-protective properties

International audienceOxidized phospholipids (OxPLs) containing enzymatically or non-enzymatically oxidized fatty acids (oxylipins) are increasingly recognized as lipid mediators involved in pathogenesis of diseases. Further understanding of structure-activity relationship and molecular mechanisms activated by OxPLs is hampered by the complexity of synthesis of individual molecular species. Although dozens of individual free oxylipins are commercially available, their attachment to the phospholipid scaffold requires relatively harsh conditions during activation of carboxy-group, which may lead to decomposition of unstable oxylipins. Furthermore, additional protection-deprotection steps are required for oxylipins containing hydroxy-groups. In this work we describe synthesis of OxPLs containing oxylipins bound at the sn-2-position via an amide-bond that is characteristic of sphingophospholipids. Activation of oxylipins and attachment to the phospholipid scaffold are performed under mild conditions and characterized by high yield. Hydroxy-groups of oxylipins do not interfere with reactions and therefore no protection/deprotection steps are needed. In order to prevent oxylipin migration, a fatty acid residue at the sn-1 was bound through an alkyl bond, which is a common bond present in a large proportion of naturally occurring phospholipids. An additional advantage of combining alkyl and amide bonds in a single phospholipid molecule is that both types of bonds are phospholipase A1/A2-resistant, which may be expected to improve biological stability of OxPLs and thus simplify analysis of their effects. As proof of principle, several alkyl-amide oxidized phosphatidylcholines (OxPCs) containing either linear or prostane ring oxylipins have been synthesized. Importantly, we show here that alkyl-amide-OxPCs demonstrated biological activities similar to those of di-acyl-OxPCs. Alkyl-amide-OxPCs inhibited pro-inflammatory action of LPS and increased endothelial cellular barrier in vitro and in mouse models. The effects of alkyl-amide and di-acyl-OxPCs developed in a similar range of concentrations. We hypothesize that alkyl-amide-OxPLs may become a useful tool for deeper analysis of the structure-activity relationship of OxPLs

Methionine oxidation in the NirA-NES defines subcellular localization and activity.

<p><b>(A)</b> NirA-GFP sub-cellular localization in different strains under non-induced (NI, 3 mM arginine), non-induced plus n-octylamine (NIo, 3 mM arginine plus 10 mM n-octylamine), induced (IND, 10 mM nitrate) and induced plus n-octylamine (INDo, 10 mM nitrate plus 10 mM n-octylamine) conditions. Pictures were taken two minutes after addition of the respective compounds. All strains harbour the Leptomycin B (LMB) sensitive allele <i>kapK</i>1. Leptomycin B sensitivity enables testing if loss of nuclear accumulation is related to/dependent on exportin (KapK) function. Except strain <i>kapK</i>1 NirA^M169A, which carries a Met to Ala substitution within the NES sequence, all strains carry a NirA-GFP wild type construct. Scale bars refer to 5 μm. <b>(B)</b> Effect of the <i>fmoB</i> deletion on <i>niaD</i> transcription. Strains were grown under standard conditions and transferred for 20 minutes to the media as indicated and described in panel A. Signal intensities were calculated with ImageQuant (Molecular Dynamics) and <i>niaD</i> expression was normalized to <i>18S</i> rRNA. <b>(C)</b> Effect of nitrogen source and n-octylamine on NirA protein stability. Whole cell extracts of a FLAG-tagged NirA strain were analysed by Western blot. The strain was grown for 16 hours under non induced conditions with 0.2% fructose and 3 mM arginine as nitrogen source; NI. The cultures where subsequently treated with 2 mM n-octylamine, NIo; 10 mM nitrate, IND; and 10 mM nitrate plus 2 mM n-octylamine, INDo. Specific signals were normalized against actin (ActA).</p

Overview of correlations between NES sequences, oxidation status of M169 (red/ox), NirA-GFP subcellular localization and NirA transcriptional activity in different genetic backgrounds of strains grown under non-inducing or inducing conditions.

<p>(n.a., not applicable, n.d., not determined)</p><p>Overview of correlations between NES sequences, oxidation status of M169 (red/ox), NirA-GFP subcellular localization and NirA transcriptional activity in different genetic backgrounds of strains grown under non-inducing or inducing conditions.</p

Interaction of KapK with the NirA-NES is sensitive to methionine oxidation.

<p><b>(A)</b> Western blot using S-tag antibody shows <i>in vitro</i> interaction of KapK with synthetic NES peptide variants containing wild type or mutated amino acid sequences. M^ox, NES peptide carrying methionine 169 sulfoxide; M, NES peptide carrying reduced methionine 169; M169A, NES peptide carrying a M169A substitution, L172A_L174A, NES peptide carrying a double L172A, L174A mutation. Signal densities reflect the interaction strengths between biotinylated NES peptides, immobilized on streptavidin sepharose, and KapK-Stag in non-induced cell-free extracts. <b>(B)</b> Western blot of co-IP experiments showing that <i>in vivo</i> NirA-KapK interaction is sensitive to the thiol redox status and to the presence of the C-terminal activation domain. Co-immunoprecipitation of cell-free extracts prepared from a strain co-expressing FLAG-NirA and KapK-S-tag. Extracts were prepared from cells grown under non-induced conditions (NI, 3 mM arginine) or induced conditions (IND, 10 mM NO₃^-). The extracts were prepared either in the presence (+), or absence (-) of 5 mM DTT. Input of pre-cleared cell-free extracts of all strains is shown in the upper panel. Elutions after the Co-IPs were separated on SDS-PAGE and equal loading of the co-IP resin with the S-tag capture Ab was verified by probing with the secondary Ab against the IgG heavy chain (~52 kDa) of the anti-S-tag antibody. A 70 kDa band of a proteolytically processed form of FLAG-NirA lacking around 30 kDa at the C-terminus is present.</p