DiagnoMass: A proteomics hub for pinpointing discriminative spectral clusters.

MOTIVATION: There are several well-established paradigms for identifying and pinpointing discriminative peptides/proteins using shotgun proteomic data; examples are peptide-spectrum matching, de novo sequencing, open searches, and even hybrid approaches. Such an arsenal of complementary paradigms can provide deep data coverage, albeit some unidentified discriminative peptides remain. RESULTS: We present DiagnoMass, software tool that groups similar spectra into spectral clusters and then shortlists those clusters that are discriminative for biological conditions. DiagnoMass then communicates with proteomic tools to attempt the identification of such clusters. We demonstrate the effectiveness of DiagnoMass by analyzing proteomic data from Escherichia coli, Salmonella, and Shigella, listing many high-quality discriminative spectral clusters that had thus far remained unidentified by widely adopted proteomic tools. DiagnoMass can also classify proteomic profiles. We anticipate the use of DiagnoMass as a vital tool for pinpointing biomarkers. AVAILABILITY: DiagnoMass and related documentation, including a usage protocol, are available at http://www.diagnomass.com.

PD-1/PD-L1 blockade abrogates a dysfunctional innate-adaptive immune axis in critical ß-coronavirus disease.

Severe COVID-19 is associated with hyperinflammation and weak T cell responses against SARS-CoV-2. However, the links between those processes remain partially characterized. Moreover, whether and how therapeutically manipulating T cells may benefit patients are unknown. Our genetic and pharmacological evidence demonstrates that the ion channel TMEM176B inhibited inflammasome activation triggered by SARS-CoV-2 and SARS-CoV-2-related murine ß-coronavirus. Tmem176b-/- mice infected with murine ß-coronavirus developed inflammasome-dependent T cell dysfunction and critical disease, which was controlled by modulating dysfunctional T cells with PD-1 blockers. In critical COVID-19, inflammasome activation correlated with dysfunctional T cells and low monocytic TMEM176B expression, whereas PD-L1 blockade rescued T cell functionality. Here, we mechanistically link T cell dysfunction and inflammation, supporting a cancer immunotherapy to reinforce T cell immunity in critical ß-coronavirus disease.

Decreased proteasomal cleavage at nitrotyrosine sites in proteins and peptides.

Removal of moderately oxidized proteins is mainly carried out by the proteasome, while highly modified proteins are no longer degradable. However, in the case of proteins modified by nitration of tyrosine residues to 3-nitrotyrosine (NO2Y), the role of the proteasome remains to be established. For this purpose, degradation assays and mass spectrometry analyses were performed using isolated proteasome and purified fractions of native cytochrome c (Cyt c) and tyrosine nitrated proteoforms (NO2Y74-Cyt c and NO2Y97-Cyt c). While Cyt c treated under mild conditions with hydrogen peroxide was preferentially degraded by the proteasome, NO2Y74- and NO2Y97-Cyt c species did not show an increased degradation rate with respect to native Cyt c. Peptide mapping analysis confirmed a decreased chymotrypsin-like cleavage at C-terminal of NO2Y sites within the protein, with respect to unmodified Y residues. Additionally, studies with the proteasome substrate suc-LLVY-AMC (Y-AMC) and its NO2Y-containing analog, suc-LLVNO2Y-AMC (NO2Y-AMC) were performed, both using isolated 20S-proteasome and astrocytoma cell lysates as the proteasomal source. Comparisons of both substrates showed a significantly decreased proteasome activity towards NO2Y-AMC. Moreover, NO2Y-AMC, but not Y-AMC degradation rates, were largely diminished by increasing the reaction pH, suggesting an inhibitory influence of the additional negative charge contained in NO2Y-AMC secondary to nitration. The mechanism of slowing of proteasome activity in NO2Y-contaning peptides was further substantiated in studies using the phenylalanine and nitro-phenylalanine peptide analog substrates. Finally, degradation rates of Y-AMC and NO2Y-AMC with proteinase K were the same, demonstrating the selective inability of the proteasome to readily cleave at nitrotyrosine sites. Altogether, data indicate that the proteasome has a decreased capability to cleave at C-terminal of NO2Y residues in proteins with respect to the unmodified residues, making this a possible factor that decreases the turnover of oxidized proteins, if they are not unfolded, and facilitating the accumulation of nitrated proteins.

The protein Deleted in Breast Cancer-1 (DBC1) regulates vascular response and formation of aortic dissection during Angiotensin II infusion.

Cardiovascular diseases are among the main causes of morbimortality in the adult population. Among them, hypertension is a leading cause for stroke, heart disease and kidney failure. Also, as a result of arterial wall weakness, hypertension can lead to the development of dissecting aortic aneurysms, a rare but often fatal condition if not readily treated. In this work, we investigated the role of DBC1 in the regulation of vascular function in an ANGII-induced hypertension mouse model. We found that WT and DBC1 KO mice developed hypertension in response to ANGII infusion. However, DBC1 KO mice showed increased susceptibility to develop aortic dissections. The effect was accompanied by upregulation of vascular remodeling factors, including MMP9 and also VEGF. Consistent with this, we found decreased collagen deposition and elastic fiber fragmentation, suggesting that increased expression of MMPs in DBC1 KO mice weakens the arterial wall, promoting the formation of aortic dissections during treatment with ANGII. Finally, DBC1 KO mice had reduced cell proliferation in the intima-media layer in response to ANGII, paralleled with an impairment to increase wall thickness in response to hypertension. Furthermore, VSMC purified from DBC1 KO mice showed impaired capacity to leave quiescence, confirming the in vivo results. Altogether, our results show for the first time that DBC1 regulates vascular response and function during hypertension and protects against vascular injury. This work also brings novel insights into the molecular mechanisms of the development of aortic dissections.

Proteomic analysis of Saccharomyces cerevisiae to study the effects of red wine polyphenols on oxidative stress.

Understanding the molecular mechanisms underlying the "French paradox" has contributed to a growing interest in the investigation of the biological activity of red wine polyphenols (RWP). The main goal of this research is to provide valuable information on how RWP could exert their biological action at the cellular level. So, we report a proteomic analysis of S. cerevisiae exposed to both pro-oxidant (H2O2) and antioxidant (wine) agents. Cellular proteome analysis shows that RWP modify the level of certain proteins. Under both normal conditions (Wine treatment) and oxidative stress situations (Wine + H2O2 treatment), the proteins involved in the metabolism and biosynthesis of biomolecules were down-regulated, while one ribosomal protein was up-regulated, probably performing its ribosome-independent functions, and so contributing to the stress defense system. Considering this action mechanism, we suggest that RWP may be acting as mild pro-oxidants and, therefore, exerting a hormetic effect that leads to the strengthening of cells' antioxidant capacity.

A Focused Library of NO-Donor Compounds with Potent Antiproliferative Activity Based on Green Multicomponent Reactions.

Cancer is the second leading cause of death worldwide. Herein, a strategy to quickly and efficiently identify novel lead compounds to develop anticancer agents, using green multicomponent reactions followed by antiproliferative activity and structure-activity relationship studies, is described. A second-generation focused library of nitric oxide-releasing compounds was prepared by microwave-assisted Passerini and Ugi reactions. Nearly all compounds displayed potent antiproliferative activities against a panel of human solid tumor cell lines, with 1-phenyl-1-[(tert-butylamino)carbonyl]methyl 3-[(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N2 -oxide)oxy]benzoate (4 k) and N-[1-(tert-butylaminocarbonyl)-1-phenylmethyl]-N-(4-methylphenyl)-3-(3-phenylsulfonyl-[1,2,5]oxadiazol-4-yl N2 -oxide)oxyphenyl carboxamide (6 d) exhibiting the strongest activity on SW1573 lung cell line (GI50 =110 and 21 nm) with selectivity indices of 70 and 470, respectively. Preliminary mechanistic studies suggest a relationship between NO release and antiproliferative activity. Our strategy allowed the rapid identification of at least two molecules as future candidates for the development of potent antitumor drugs.

Electrophiles modulate glutathione reductase activity via alkylation and upregulation of glutathione biosynthesis.

Cells evolved robust homeostatic mechanisms to protect against oxidation or alkylation by electrophilic species. Glutathione (GSH) is the most abundant intracellular thiol, protects cellular components from oxidation and is maintained in a reduced state by glutathione reductase (GR). Nitro oleic acid (NO2-OA) is an electrophilic fatty acid formed under digestive and inflammatory conditions that both reacts with GSH and induces its synthesis upon activation of Nrf2 signaling. The effects of NO2-OA on intracellular GSH homeostasis were evaluated. In addition to upregulation of GSH biosynthesis, we observed that NO2-OA increased intracellular GSSG in an oxidative stress-independent manner. NO2-OA directly inhibited GR in vitro by covalent modification of the catalytic Cys61, with kon of (3.45 ± 0.04) × 103 M-1 s-1, koff of (4.4 ± 0.4) × 10-4 s-1, and Keq of (1.3 ± 0.1) × 10-7 M. Akin to NO2-OA, the electrophilic Nrf2 activators bardoxolone-imidazole (CDDO-Im), bardoxolone-methyl (CDDO-Me) and dimethyl fumarate (DMF) also upregulated GSH biosynthesis while promoting GSSG accumulation, but without directly inhibiting GR activity. In vitro assays in which GR was treated with increasing GSH concentrations and GSH depletion experiments in cells revealed that GR activity is finely regulated via product inhibition, an observation further supported by theoretical (kinetic modeling of cellular GSSG:GSH levels) approaches. Together, these results describe two independent mechanisms by which electrophiles modulate the GSH/GSSG couple, and provide a novel conceptual framework to interpret experimentally determined values of GSH and GSSG.

A novel nitroalkene-α-tocopherol analogue inhibits inflammation and ameliorates atherosclerosis in Apo E knockout mice.

BACKGROUND AND PURPOSE: Atherosclerosis is characterized by chronic low-grade inflammation with concomitant lipid accumulation in the arterial wall. Anti-inflammatory and anti-atherogenic properties have been described for a novel class of endogenous nitroalkenes (nitrated-unsaturated fatty acids), formed during inflammation and digestion/absorption processes. The lipid-associated antioxidant α-tocopherol is transported systemically by LDL particles including to the atheroma lesions. To capitalize on the overlapping and complementary salutary properties of endogenous nitroalkenes and α-tocopherol, we designed and synthesized a novel nitroalkene-α-tocopherol analogue (NATOH) to address chronic inflammation and atherosclerosis, particularly at the lesion sites. EXPERIMENTAL APPROACH: We synthesized NATOH, determined its electrophilicity and antioxidant capacity and studied its effects over pro-inflammatory and cytoprotective pathways in macrophages in vitro. Moreover, we demonstrated its incorporation into lipoproteins and tissue both in vitro and in vivo, and determined its effect on atherosclerosis and inflammatory responses in vivo using the Apo E knockout mice model. KEY RESULTS: NATOH exhibited similar antioxidant capacity to α-tocopherol and, due to the presence of the nitroalkenyl group, like endogenous nitroalkenes, it exerted electrophilic reactivity. NATOH was incorporated in vivo into the VLDL/LDL lipoproteins particles to reach the atheroma lesions. Furthermore, oral administration of NATOH down-regulated NF-κB-dependent expression of pro-inflammatory markers (including IL-1ß and adhesion molecules) and ameliorated atherosclerosis in Apo E knockout mice. CONCLUSIONS AND IMPLICATIONS: In toto, the data demonstrate a novel pharmacological strategy for the prevention of atherosclerosis based on a creative, natural and safe drug delivery system of a non-conventional anti-inflammatory compound (NATOH) with significant potential for clinical application.

Electrophilic nitroalkene-tocopherol derivatives: synthesis, physicochemical characterization and evaluation of anti-inflammatory signaling responses.

Inflammation plays a major role in the onset and development of chronic non-communicable diseases like obesity, cardiovascular diseases and cancer. Combined, these diseases represent the most common causes of death worldwide, thus development of novel pharmacological approaches is crucial. Electrophilic nitroalkenes derived from fatty acids are formed endogenously and exert anti-inflammatory actions by the modification of proteins involved in inflammation signaling cascades. We have developed novel nitroalkenes derived from α-tocopherol aiming to increase its salutary actions by adding anti-inflammatory properties to a well-known nutraceutical. We synthesized and characterized an α-tocopherol-nitroalkene (NATOH) and two hydrosoluble analogues derived from Trolox (NATxME and NATx0). We analyzed the kinetics of the Michael addition reaction of these compounds with thiols in micellar systems aiming to understand the effect of hydrophobic partition on the reactivity of nitroalkenes. We studied NATxME in vitro showing it exerts non-conventional anti-inflammatory responses by inducing Nrf2-Keap1-dependent gene expression and inhibiting the secretion of NF-κB dependent pro-inflammatory cytokines. NATxME was also effective in vivo, inhibiting neutrophil recruitment in a zebrafish model of inflammation. This work lays the foundation for the rational design of a new therapeutic strategy for the prevention and treatment of metabolic and inflammation-related diseases.

A green multicomponent synthesis of tocopherol analogues with antiproliferative activities.

A one-pot efficient, practical and eco-friendly synthesis of tocopherol analogues has been developed using water or solvent free conditions via Passerini and Ugi multicomponent reactions. These reactions can be optimized using microwave irradiation or ultrasound as the energy source. Accordingly, a small library of 30 compounds was prepared for biological tests. The evaluation of the antiproliferative activity in the human solid tumor cell lines A549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (lung), T-47D (breast), and WiDr (colon) provided lead compounds with GI50 values between 1 and 5 µM. A structure-activity relationship is also discussed. One of the studied compounds comes up as a future candidate for the development of potent tocopherol-mimetic therapeutic agents for cancer.

Characterisation of Antigen B Protein Species Present in the Hydatid Cyst Fluid of Echinococcus canadensis G7 Genotype.

The larva of cestodes belonging to the Echinococcus granulosus sensu lato (s.l.) complex causes cystic echinococcosis (CE). It is a globally distributed zoonosis with significant economic and public health impact. The most immunogenic and specific Echinococcus-genus antigen for human CE diagnosis is antigen B (AgB), an abundant lipoprotein of the hydatid cyst fluid (HF). The AgB protein moiety (apolipoprotein) is encoded by five genes (AgB1-AgB5), which generate mature 8 kDa proteins (AgB8/1-AgB8/5). These genes seem to be differentially expressed among Echinococcus species. Since AgB immunogenicity lies on its protein moiety, differences in AgB expression within E. granulosus s.l. complex might have diagnostic and epidemiological relevance for discriminating the contribution of distinct species to human CE. Interestingly, AgB2 was proposed as a pseudogene in E. canadensis, which is the second most common cause of human CE, but proteomic studies for verifying it have not been performed yet. Herein, we analysed the protein and lipid composition of AgB obtained from fertile HF of swine origin (E. canadensis G7 genotype). AgB apolipoproteins were identified and quantified using mass spectrometry tools. Results showed that AgB8/1 was the major protein component, representing 71% of total AgB apolipoproteins, followed by AgB8/4 (15.5%), AgB8/3 (13.2%) and AgB8/5 (0.3%). AgB8/2 was not detected. As a methodological control, a parallel analysis detected all AgB apolipoproteins in bovine fertile HF (G1/3/5 genotypes). Overall, E. canadensis AgB comprised mostly AgB8/1 together with a heterogeneous mixture of lipids, and AgB8/2 was not detected despite using high sensitivity proteomic techniques. This endorses genomic data supporting that AgB2 behaves as a pseudogene in G7 genotype. Since recombinant AgB8/2 has been found to be diagnostically valuable for human CE, our findings indicate that its use as antigen in immunoassays could contribute to false negative results in areas where E. canadensis circulates. Furthermore, the presence of anti-AgB8/2 antibodies in serum may represent a useful parameter to rule out E. canadensis infection when human CE is diagnosed.

Nitration transforms a sensitive peroxiredoxin 2 into a more active and robust peroxidase.

Peroxiredoxins (Prx) are efficient thiol-dependent peroxidases and key players in the mechanism of H2O2-induced redox signaling. Any structural change that could affect their redox state, oligomeric structure, and/or interaction with other proteins could have a significant impact on the cascade of signaling events. Several post-translational modifications have been reported to modulate Prx activity. One of these, overoxidation of the peroxidatic cysteine to the sulfinic derivative, inactivates the enzyme and has been proposed as a mechanism of H2O2 accumulation in redox signaling (the floodgate hypothesis). Nitration of Prx has been reported in vitro as well as in vivo; in particular, nitrated Prx2 was identified in brains of Alzheimer disease patients. In this work we characterize Prx2 tyrosine nitration, a post-translational modification on a noncatalytic residue that increases its peroxidase activity and its resistance to overoxidation. Mass spectrometry analysis revealed that treatment of disulfide-oxidized Prx2 with excess peroxynitrite renders mainly mononitrated and dinitrated species. Tyrosine 193 of the YF motif at the C terminus, associated with the susceptibility toward overoxidation of eukaryotic Prx, was identified as nitrated and is most likely responsible for the protection of the peroxidatic cysteine against oxidative inactivation. Kinetic analyses suggest that tyrosine nitration facilitates the intermolecular disulfide formation, transforming a sensitive Prx into a robust one. Thus, tyrosine nitration appears as another mechanism to modulate these enzymes in the complex network of redox signaling.

Structural and molecular basis of the peroxynitrite-mediated nitration and inactivation of Trypanosoma cruzi iron-superoxide dismutases (Fe-SODs) A and B: disparate susceptibilities due to the repair of Tyr35 radical by Cys83 in Fe-SODB through intramolecular electron transfer.

Trypanosoma cruzi, the causative agent of Chagas disease, contains exclusively iron-dependent superoxide dismutases (Fe-SODs) located in different subcellular compartments. Peroxynitrite, a key cytotoxic and oxidizing effector biomolecule, reacted with T. cruzi mitochondrial (Fe-SODA) and cytosolic (Fe-SODB) SODs with second order rate constants of 4.6 ± 0.2 × 10(4) M(-1) s(-1) and 4.3 ± 0.4 × 10(4) M(-1) s(-1) at pH 7.4 and 37 °C, respectively. Both isoforms are dose-dependently nitrated and inactivated by peroxynitrite. Susceptibility of T. cruzi Fe-SODA toward peroxynitrite was similar to that reported previously for Escherichia coli Mn- and Fe-SODs and mammalian Mn-SOD, whereas Fe-SODB was exceptionally resistant to oxidant-mediated inactivation. We report mass spectrometry analysis indicating that peroxynitrite-mediated inactivation of T. cruzi Fe-SODs is due to the site-specific nitration of the critical and universally conserved Tyr(35). Searching for structural differences, the crystal structure of Fe-SODA was solved at 2.2 Å resolution. Structural analysis comparing both Fe-SOD isoforms reveals differences in key cysteines and tryptophan residues. Thiol alkylation of Fe-SODB cysteines made the enzyme more susceptible to peroxynitrite. In particular, Cys(83) mutation (C83S, absent in Fe-SODA) increased the Fe-SODB sensitivity toward peroxynitrite. Molecular dynamics, electron paramagnetic resonance, and immunospin trapping analysis revealed that Cys(83) present in Fe-SODB acts as an electron donor that repairs Tyr(35) radical via intramolecular electron transfer, preventing peroxynitrite-dependent nitration and consequent inactivation of Fe-SODB. Parasites exposed to exogenous or endogenous sources of peroxynitrite resulted in nitration and inactivation of Fe-SODA but not Fe-SODB, suggesting that these enzymes play distinctive biological roles during parasite infection of mammalian cells.

Inhibition of Mycobacterium tuberculosis PknG by non-catalytic rubredoxin domain specific modification: reaction of an electrophilic nitro-fatty acid with the Fe-S center.

PknG from Mycobacterium tuberculosis is a Ser/Thr protein kinase that regulates key metabolic processes within the bacterial cell as well as signaling pathways from the infected host cell. This multidomain protein has a conserved canonical kinase domain with N- and C-terminal flanking regions of unclear functional roles. The N-terminus harbors a rubredoxin-like domain (Rbx), a bacterial protein module characterized by an iron ion coordinated by four cysteine residues. Disruption of the Rbx-metal binding site by simultaneous mutations of all the key cysteine residues significantly impairs PknG activity. This encouraged us to evaluate the effect of a nitro-fatty acid (9- and 10-nitro-octadeca-9-cis-enoic acid; OA-NO2) on PknG activity. Fatty acid nitroalkenes are electrophilic species produced during inflammation and metabolism that react with nucleophilic residues of target proteins (i.e., Cys and His), modulating protein function and subcellular distribution in a reversible manner. Here, we show that OA-NO2 inhibits kinase activity by covalently adducting PknG remote from the catalytic domain. Mass spectrometry-based analysis established that cysteines located at Rbx are the specific targets of the nitroalkene. Cys-nitroalkylation is a Michael addition reaction typically reverted by thiols. However, the reversible OA-NO2-mediated nitroalkylation of the kinase results in an irreversible inhibition of PknG. Cys adduction by OA-NO2 induced iron release from the Rbx domain, revealing a new strategy for the specific inhibition of PknG. These results affirm the relevance of the Rbx domain as a target for PknG inhibition and support that electrophilic lipid reactions of Rbx-Cys may represent a new drug strategy for specific PknG inhibition.

Characterisation of the native lipid moiety of Echinococcus granulosus antigen B.

Antigen B (EgAgB) is the most abundant and immunogenic antigen produced by the larval stage (metacestode) of Echinococcus granulosus. It is a lipoprotein, the structure and function of which have not been completely elucidated. EgAgB apolipoprotein components have been well characterised; they share homology with a group of hydrophobic ligand binding proteins (HLBPs) present exclusively in cestode organisms, and consist of different isoforms of 8-kDa proteins encoded by a polymorphic multigene family comprising five subfamilies (EgAgB1 to EgAgB5). In vitro studies have shown that EgAgB apolipoproteins are capable of binding fatty acids. However, the identity of the native lipid components of EgAgB remains unknown. The present work was aimed at characterising the lipid ligands bound to EgAgB in vivo. EgAgB was purified to homogeneity from hydatid cyst fluid and its lipid fraction was extracted using chloroform∶methanol mixtures. This fraction constituted approximately 40-50% of EgAgB total mass. High-performance thin layer chromatography revealed that the native lipid moiety of EgAgB consists of a variety of neutral (mainly triacylglycerides, sterols and sterol esters) and polar (mainly phosphatidylcholine) lipids. Gas-liquid chromatography analysis showed that 16∶0, 18∶0 and 18∶1(n-9) are the most abundant fatty acids in EgAgB. Furthermore, size exclusion chromatography coupled to light scattering demonstrated that EgAgB comprises a population of particles heterogeneous in size, with an average molecular mass of 229 kDa. Our results provide the first direct evidence of the nature of the hydrophobic ligands bound to EgAgB in vivo and indicate that the structure and composition of EgAgB lipoprotein particles are more complex than previously thought, resembling high density plasma lipoproteins. Results are discussed considering what is known on lipid metabolism in cestodes, and taken into account the Echinococcus spp. genomic information regarding both lipid metabolism and the EgAgB gene family.

Phagocyte-specific S100 proteins in the local response to the Echinococcus granulosus larva.

Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory control. However, various degrees of chronic granulomatous inflammation are also observed, reaching a high point in infection of cattle by the most prevalent parasite strain worldwide, which is not well adapted to this host species. In this context, epithelioid and multinucleated giant macrophages surround the parasite, and the secreted products of these cells often associate with the larval wall. The phagocyte-specific S100 proteins, S100A8, S100A9 and S100A12, are important non-conventionally secreted amplifiers of inflammatory responses. We have analysed by proteomics and immunohistochemistry the presence of these proteins at the E. granulosus larva-host interface. We found that, in the context of inflammatory control as observed in human infections, the S100 proteins are not abundant, but S100A9 and S100A8 can be expressed by eosinophils distal to the parasite. In the granulomatous inflammation context as observed in cattle infections, we found that S100A12 is one of the most abundant host-derived, parasite-associated proteins, while S100A9 and S100A8 are not present at similarly high levels. As expected, S100A12 derives mostly from the epithelioid and multinucleated giant cells. S100A12, as well as cathepsin K and matrix metalloproteinase-9, also expressed by E. granulosus-elicited epithelioid cells, are connected to the Th17 arm of immunity, which may therefore be involved in this granulomatous response.

Adenosine triphosphate-dependent calcium signaling during ventilator-induced lung injury is amplified by hypercapnia.

Adenosine triphosphate (ATP) is released by alveolar epithelial cells during ventilator-induced lung injury (VILI) and regulates fluid transport across epithelia. High CO(2) levels are observed in patients with "permissive hypercapnia," which inhibits alveolar fluid reabsorption (AFR) in alveolar epithelial cells. The authors set out to determine whether VILI affects AFR and whether the purinergic pathway is modulated in cells exposed to hypercapnia. Control group was compared against VILI (tidal volume [Vt] = 35 mL/kg, zero positive end-expiratory pressure [PEEP]) and protective ventilation (Vt = 6 mL/kg, PEEP = 10 cm H(2)O) groups. Lung mechanics, histology, and AFR were evaluated. Alveolar epithelial cells (AECs) were loaded with Fura 2-AM to measure intracellular calcium in the presence ATP (10 µM) at 5% or 10% CO(2) as compared with baseline. High tidal volume ventilation impairs lung mechanics and AFR. Hypercapnia (HC) increases intracellular calcium levels in response to ATP stimulation. HC + ATP is the most detrimental combination decreasing AFR. Purinergic signaling in AECs is modulated by high CO(2) levels via increased cytosolic calcium. The authors reason that this modulation may play a role in the impairment of alveolar epithelial functions induced by hypercapnia.

Electrophilic fatty acids regulate matrix metalloproteinase activity and expression.

Nitro-fatty acids (NO(2)-FA) are electrophilic signaling mediators formed by reactions of nitric oxide and nitrite. NO(2)-FA exert anti-inflammatory signaling actions through post-translational protein modifications. We report that nitro-oleic acid (OA-NO(2)) stimulates proMMP-7 and proMMP-9 proteolytic activity via adduction of the conserved cysteine switch domain thiolate. Biotin-labeled OA-NO(2) showed this adduction occurs preferentially with latent forms of MMP, confirming a role for thiol alkylation by OA-NO(2) in MMP activation. In addition to regulating pro-MMP activation, MMP expression was modulated by OA-NO(2) via activation of peroxisome proliferator-activated receptor-γ. MMP-9 transcription was decreased in phorbol 12-myristate 13-acetate-stimulated THP-1 macrophages to an extent similar to that induced by the peroxisome proliferator-activated receptor-γ agonist Rosiglitazone. This was affirmed using a murine model of atherosclerosis, ApoE(-/-) mice, where in vivo OA-NO(2) administration suppressed MMP expression in atherosclerotic lesions. These findings reveal that electrophilic fatty acid derivatives can serve as effectors during inflammation, first by activating pro-MMP proteolytic activity via alkylation of the cysteine switch domain, and then by transcriptionally inhibiting MMP expression, thereby limiting the further progression of inflammatory processes.

Macrophage activation induces formation of the anti-inflammatory lipid cholesteryl-nitrolinoleate.

Nitroalkene derivatives of fatty acids act as adaptive, anti-inflammatory signalling mediators, based on their high-affinity PPARgamma (peroxisome-proliferator-activated receptor gamma) ligand activity and electrophilic reactivity with proteins, including transcription factors. Although free or esterified lipid nitroalkene derivatives have been detected in human plasma and urine, their generation by inflammatory stimuli has not been reported. In the present study, we show increased nitration of cholesteryl-linoleate by activated murine J774.1 macrophages, yielding the mononitrated nitroalkene CLNO2 (cholesteryl-nitrolinoleate). CLNO2 levels were found to increase approximately 20-fold 24 h after macrophage activation with Escherichia coli lipopolysaccharide plus interferon-gamma; this response was concurrent with an increase in the expression of NOS2 (inducible nitric oxide synthase) and was inhibited by the (*)NO (nitric oxide) inhibitor L-NAME (N(G)-nitro-L-arginine methyl ester). Macrophage (J774.1 and bone-marrow-derived cells) inflammatory responses were suppressed when activated in the presence of CLNO2 or LNO2 (nitrolinoleate). This included: (i) inhibition of NOS2 expression and cytokine secretion through PPARgamma and *NO-independent mechanisms; (ii) induction of haem oxygenase-1 expression; and (iii) inhibition of NF-kappaB (nuclear factor kappaB) activation. Overall, these results suggest that lipid nitration occurs as part of the response of macrophages to inflammatory stimuli involving NOS2 induction and that these by-products of nitro-oxidative reactions may act as novel adaptive down-regulators of inflammatory responses.

Nitro-oleic acid, a novel and irreversible inhibitor of xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) generates proinflammatory oxidants and secondary nitrating species, with inhibition of XOR proving beneficial in a variety of disorders. Electrophilic nitrated fatty acid derivatives, such as nitro-oleic acid (OA-NO2), display anti-inflammatory effects with pleiotropic properties. Nitro-oleic acid inhibits XOR activity in a concentration-dependent manner with an IC50 of 0.6 microM, limiting both purine oxidation and formation of superoxide (O2.). Enzyme inhibition by OA-NO2 is not reversed by thiol reagents, including glutathione, beta-mercaptoethanol, and dithiothreitol. Structure-function studies indicate that the carboxylic acid moiety, nitration at the 9 or 10 olefinic carbon, and unsaturation is required for XOR inhibition. Enzyme turnover and competitive reactivation studies reveal inhibition of electron transfer reactions at the molybdenum cofactor accounts for OA-NO2-induced inhibition. Importantly, OA-NO2 more potently inhibits cell-associated XOR-dependent O2. production than does allopurinol. Combined, these data establish a novel role for OA-NO2 in the inhibition of XOR-derived oxidant formation.