Tânia Melo

Lipoxidation adducts with peptides and proteins: deleterious modifications or signaling mechanisms?

Protein lipoxidation refers to the modification by electrophilic lipid oxidation products to form covalent adducts, which for many years has been considered as a deleterious consequence of oxidative stress. Oxidized lipids or phospholipids containing carbonyl moieties react readily with lysine to form Schiff bases; alternatively, oxidation products containing α,β-unsaturated moieties are susceptible to nucleophilic attack by cysteine, histidine or lysine residues to yield Michael adducts, overall corresponding to a large number of possible protein adducts. The most common detection methods for lipoxidized proteins take advantage of the presence of reactive carbonyl groups to add labels, or use antibodies. These methods have limitations in terms of specificity and identification of the modification site. The latter question is satisfactorily addressed by mass spectrometry, which enables the characterization of the adduct structure. This has allowed the identification of lipoxidized proteins in physiological and pathological situations. While in many cases lipoxidation interferes with protein function, causing inhibition of enzymatic activity and increased immunogenicity, there are a small number of cases where lipoxidation results in gain of function or activity. For certain proteins lipoxidation may represent a form of redox signaling, although more work is required to confirm the physiological relevance and mechanisms of such processes. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Cardiolipin Profile Changes are Associated to the Early Synaptic Mitochondrial Dysfunction in Alzheimer's Disease

Brain mitochondria are fundamental to maintaining healthy functional brains, and their dysfunction is involved in age-related neurodegenerative disorders such as Alzheimers disease (AD). In this study, we conducted a research on how both non-synaptic and synaptic mitochondrial functions are compromised at an early stage of AD-like pathologies and their correlation with putative changes on membranes lipid profile, using 3 month-old nontransgenic and 3xTg-AD mice, a murine model of experimental AD. Bioenergetic dysfunction in 3xTg-AD brains is evidenced by a decrease of brain ATP levels resulting, essentially, from synaptic mitochondria functionality disruption as indicated by declined respiratory control ratio associated with a 50% decreased complex I activity. Lipidomics studies revealed that synaptic bioenergetic deficit of 3xTg-AD brains is accompanied by alterations in the phospholipid composition of synaptic mitochondrial membranes, detected either in phospholipid class distribution or in the phospholipids molecular profile. Globally, diacyl- and lyso-phosphatidylcholine lipids increase while ethanolamine plasmalogens and cardiolipins content drops in relation to nontransgenic background. However, the main lipidomic mark of 3xTg-AD brains is that cardiolipin cluster-organized profile is lost in synaptic mitochondria due to a decline of the most representative molecular species. In contrast to synaptic mitochondria, results support the idea that non-synaptic mitochondria function is preserved at the age of 3 months. Although the genetically construed 3xTg-AD mouse model does not represent the most prevalent form of AD in humans, the present study provides insights into the earliest biochemical events in AD brain, connecting specific lipidomic changes with synaptic bioenergetic deficit that may contribute to the progressive synapses loss and the neurodegenerative process that characterizes AD.

Photodynamic oxidation of Escherichia coli membrane phospholipids: new insights based on lipidomics

RATIONALE The irreversible oxidation of biological molecules, such as lipids, can be achieved with a photosensitizing agent and subsequent exposure to light, in the presence of molecular oxygen. Although lipid peroxidation is an important toxicity mechanism in bacteria, the alterations caused by the photodynamic therapy on bacterial phospholipids are still unknown. In this work, we studied the photodynamic oxidation of Escherichia coli membrane phospholipids using a lipidomic approach. METHODS E. coli ATCC 25922 were irradiated for 90 min with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of a tricationic porphyrin [(5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. Lipids were extracted and separated by thin-layer chromatography. Phospholipid classes were quantified by phosphorus assay and analyzed by electrospray ionization tandem mass spectrometry. Fatty acids were analyzed by gas chromatography. Quantification of lipid hydroperoxides was performed by FOX2 assay. Analysis of the photodynamic oxidation of a phospholipid standard was also performed. RESULTS Our approach allowed us to see that the photodynamic treatment induced the formation of a high amount of lipid hydroperoxides in the E. coli lipid extract. Quantification of fatty acids revealed a decrease in the unsaturated C16:1 and C18:1 species suggesting that oxidative modifications were responsible for their variation. It was also observed that photosensitization induced the oxidation of phosphatidylethanolamines with C16:1, C18:1 and C18:2 fatty acyl chains, with formation of hydroxy and hydroperoxy derivatives. CONCLUSIONS Membrane phospholipids of E. coli are molecular targets of the photodynamic effect induced by Tri-Py(+) -Me-PF. The overall change in the relative amount of unsaturated fatty acids and the formation of PE hydroxides and hydroperoxides evidence the damages in bacterial phospholipids caused by this lethal treatment.

Tacrine and its analogues impair mitochondrial function and bioenergetics : a lipidomic analysis in rat brain

J. Neurochem. (2012) 120, 993–1013.

Lipidomic characterization of streptozotocin-induced heart mitochondrial dysfunction.

Myocardial mitochondria dysfunction seems to represent an important pathogenic factor underlying cardiomyopathy, a common complication of type 1 diabetes mellitus (T1DM). Despite significant progress in the understanding of the molecular mechanisms of mitochondrial function in the heart, the interplay between phospholipids and membrane proteins of this organelle is still poorly comprehended. Using a well-characterized animal model of T1DM obtained by the administration of streptozotocin, phospholipid profiling of isolated mitochondria was performed using MS-based approaches, which was analyzed together with oxidative phosphorylation (OXPHOS) complexes activities and their susceptibility to oxidation, and the expression of cytochrome c, the uncoupling protein UCP-3 and the mitochondrial transcription factor Tfam. Although in higher amounts, mitochondria from T1DM heart presented lower OXPHOS activity and lower transcription ability. This profile was related to phospholipid (PL) remodeling characterized by higher phosphatidylcholine levels, lower phosphatidylglycerol, phosphatidylinositol and sphingomyelin content, higher amounts of long fatty acyl side chains and increased lipid peroxidation, particularly of cardiolipin (CL). CL peroxidation was paralleled by lower cytochrome c content. Though in higher levels, UCP-3 does not seem to protect heart mitochondrial PL and membrane proteins from the oxidative damage induced by four weeks of hyperglycemia. Taken together, our data suggest that PL remodeling of heart mitochondria is an early event in T1DM pathogenesis and is related with OXPHOS dysfunction.

Photodynamic oxidation of Staphylococcus warneri membrane phospholipids: new insights based on lipidomics

RATIONALE The photodynamic process involves the combined use of light and a photosensitizer, which, in the presence of oxygen, originates cytotoxic species capable of oxidizing biological molecules, such as lipids. However, the effect of the photodynamic process in the bacterial phospholipid profile by a photosensitizer has never been reported. A lipidomic approach was used to study the photodynamic oxidation of membrane phospholipids of Staphylococcus warneri by a tricationic porphyrin [5,10,15-tris(1-methylpyridinium-4-yl)-20-(pentafluorophenyl)porphyrin triiodide, Tri-Py(+)-Me-PF]. METHODS S. warneri (10(8) colony forming units mL(-1)) was irradiated with white light (4 mW cm(-2), 21.6 J cm(-2)) in the presence of Tri-Py(+)-Me-PF (5.0 μM). Non-photosensitized bacteria were used as control (irradiated without porphyrin). After irradiation, total lipids were extracted and separated by thin-layer chromatography (TLC). Isolated fractions of lipid classes were quantified by phosphorus assay and analyzed by mass spectrometry (MS): off-line TLC/ESI-MS, hydrophilic interaction (HILIC)-LC/MS and MS/MS. RESULTS The most representative classes of S. warneri phospholipids were identified as phosphatidylglycerols (PGs) and cardiolipins (CLs). Lysyl-phosphatidylglycerols (LPGs), phosphatidylethanolamines (PEs), phosphatidylcholines (PCs) and phosphatidic acids (PAs) were also identified. After photodynamic treatment, an overall increase in the relative abundance of PGs was observed as well as the appearance of new oxidized species from CLs, including hydroxy and hydroperoxy derivatives. Formation of high amounts of lipid hydroperoxides was confirmed by FOX2 assay. Photodynamic oxidation of phospholipid standards revealed the formation of hydroperoxy and dihydroperoxy derivatives, confirming the observed CL oxidized species in S. warneri. CONCLUSIONS Membrane phospholipids of S. warneri are molecular targets of the photoinactivation process induced by Tri-Py(+) -Me-PF. The overall modification in the relative amount of phospholipids and the formation of lipid hydroxides and hydroperoxides indicate the lethal damage caused to photosensitized bacterial cells.

Lipidomics of Mesenchymal Stromal Cells: Understanding the Adaptation of Phospholipid Profile in Response to Pro-Inflammatory Cytokines

Mesenchymal stromal cells (MSCs) present anti‐inflammatory properties and are being used with great success as treatment for inflammatory and autoimmune diseases. In clinical applications MSCs are subjected to a strong pro‐inflammatory environment, essential to their immunosuppressive action. Despite the wide clinical use of these cells, how MSCs exert their effect remains unclear. Several lipids are known to be involved in cells signaling and modulation of cellular functions. The aim of this paper is to examine the variation in lipid profile of MSCs under pro‐inflammatory environment, induced by the presence of tumor necrosis factor alpha (TNF‐α) and interferon gamma (IFN‐γ), using the most modern lipidomic approach. Major changes in lipid molecular profile of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), lysoPC (LPC), and sphingomyelin (SM) classes were found. No changes were observed in the phosphatidylinositol (PI) profile. The levels of PC species with shorter fatty acids (FAs), mainly C16:0, decreased under pro‐inflammatory stimuli. The level of PC(40:6) also decreased, which may be correlated with enhanced levels of LPC(18:0), which is known to be an anti‐inflammatory LPC, observed in MSCs subjected to TNF‐α and IFN‐γ. Simultaneously, the relative amounts of PC(36:1) and PC(38:4) increased. TNF‐α and IFN‐γ also enhanced the levels of PE(40:6) and decreased the levels of PE(O‐38:6). Higher expression of PS(36:1) and SM(34:0) along with a decrease in PS(38:6) levels were observed. These results indicate that lipid metabolism and signaling are modulated during MSCs activation, which suggests that lipids may be involved in MSCs functional and anti‐inflammatory activities. J. Cell. Physiol. 231: 1024–1032, 2016.

Photooxidation of glycated and non-glycated phosphatidylethanolamines monitored by mass spectrometry.

Phosphatidylethanolamines (PE) are one of the major components of cells membranes, namely in skin and in retina, that are continuously exposed to solar UV radiation being major targets of photooxidation damage. In addition, due to the presence of the free amine group, PE can also undergo glycation, in hyperglycemic conditions which may increase the susceptibility to oxidation. The aim of this study is to develop a model, based on mass spectrometry (MS) analysis, to identify photooxidative degradation of selected PE (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4) and glycated PEs due to UV irradiation. Photooxidation products were analysed by electrospray ionization MS (ESI-MS) and tandem MS (ESI-MS/MS) in positive and negative mode. Emphasis is placed in the influence of glycation in the generation of distinct photooxidation products. ESI-MS spectra of PE after UV photo-irradiation showed mainly hydroperoxy derivatives, due to oxidation of unsaturated fatty acyl chains. Glycated PE gave rise to several new photooxidation products formed due to oxidative cleavages of the glucose moiety, namely between C1 and C2, C2 and C3, and C5 and C6 of this sugar unit. These new products were identified by ESI-MS/MS in positive mode showing distinct neutral loss depending on the different structure of the polar head group. These new identified advanced glycated photooxidation products may have a deleterious role in the etiology of diabetic retinopathy and in diabetic retinal microvascular complications.

Evaluation of the interplay among the charge of porphyrinic photosensitizers, lipid oxidation and photoinactivation efficiency in Escherichia coli.

Photodynamic inactivation (PDI) is a simple and controllable method to destroy microorganisms based on the production of reactive oxygen species (ROS) (e.g., free radicals and singlet oxygen), which irreversibly oxidize microorganisms vital constituents resulting in lethal damage. This process requires the combined action of oxygen, light and a photosensitizer (PS), which absorbs and uses the energy from light to produce ROS. For a better understanding of the photoinactivation process, the knowledge on how some molecular targets are affected by PDI assumes great importance. The aim of this work was to study the relation between the number and position of positive charges on porphyrinic macrocycles and the changes observed on bacterial lipids. For that, five porphyrin derivatives, bearing one to four positive charges, already evaluated as PS on Escherichia coli inactivation, have been tested on lipid extracts from this bacterium, and also on a simple liposome model. The effects were evaluated by the quantification of lipid hydroperoxides and by analysis of the variation of fatty acyl profiles. E. coli suspensions and liposomes were irradiated with white light in the presence of each PS (5.0 μM). Afterwards, total E. coli lipids were extracted and quantified by phosphorus assay. Lipid oxidation on bacteria and on liposomes was quantified by ferrous oxidation in xylenol orange (FOX2 assay) and the analysis of the fatty acid profile was done by gas chromatography (GC). As previously observed for E. coli viability, an overall increase in the lipid hydroperoxides content, depending on the PS charge and on its distribution on the macrocycle, was observed. Analysis of the fatty acid profile has shown a decrease of the unsaturated fatty acids, corroborating the relation between lipid oxidation and PDI efficiency. Bacterial membrane phospholipids are important molecular targets of photoinactivation and the number of charges of the PS molecule, as well as their distribution, have a clear effect on the lipid oxidation and on the efficiency of PDI. The distinct extent of the formation of lipid hydroperoxy derivatives, depending on the PS used, is a good indicator of this process. The FOX2 assay allowed the detection of lipid peroxidation of E. coli membrane after PDI with all the five porphyrins, however, it was not the most appropriated method to quantify the relative lipid oxidation caused by PS with different efficiencies. The fatty acid analysis used to quantify the extent of lipid oxidation by the different PS provided better results. The same results were observed for the liposome model. Consequently, the model system based on liposomes is a fast and simple method that can be used for the screening of the efficiency of new PS, before proceeding with the more complex studies on bacterial models.

Photosensitized oxidation of phosphatidylethanolamines monitored by electrospray tandem mass spectrometry

Photodynamic therapy combines visible light and a photosensitizer (PS) in the presence of molecular oxygen to generate reactive oxygen species able to modify biological structures such as phospholipids. Phosphatidylethanolamines (PEs), being major phospholipid constituents of mammalian cells and membranes of Gram-negative bacteria, are potential targets of photosensitization. In this work, the oxidative modifications induced by white light in combination with cationic porphyrins (Tri-Py(+)-Me-PF and Tetra-Py(+)-Me) were evaluated on PE standards. Electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS) were used to identify and characterize the oxidative modifications induced in PEs (POPE: PE 16:0/18:1, PLPE: PE 16:0/18:2, PAPE: PE 16:0/20:4). Photo-oxidation products of POPE, PLPE and PAPE as hydroxy, hydroperoxy and keteno derivatives and products due to oxidation in ethanolamine polar head were identified. Hydroperoxy-PEs were found to be the major photo-oxidation products. Quantification of hydroperoxides (PE-OOH) allowed differentiating the potential effect in photodamage of the two porphyrins. The highest amounts of PE-OOH were notorious in the presence of Tri-Py(+)-Me-PF, a highly efficient PS against bacteria. The identification of these modifications in PEs is an important key point in the understanding cell damage processes underlying photodynamic therapy approaches.