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

Antioxidants ◽  
2018 ◽  
Vol 7 (9) ◽  
pp. 116 ◽  
Author(s):  
Bernd Gesslbauer ◽  
David Kuerzl ◽  
Niko Valpatic ◽  
Valery Bochkov
Keyword(s):  
Oxidative Stress ◽  
Mass Spectrometry ◽  
Electrophoretic Mobility ◽  
Complex Mixture ◽  
Peptide Sequencing ◽  
Covalent Modification ◽  
Oxidation Products ◽  
Simple Method ◽  
Lipid Oxidation Products ◽  
Modified Proteins

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.

scholarly journals Interactions of Gliotoxin with Protein Cysteines

2021 ◽  
Author(s):  
◽  
Rana Fathizargaran
Keyword(s):  
Mass Spectrometry ◽  
Creatine Kinase ◽  
Mammalian Cells ◽  
Protein Modification ◽  
Sulfur Oxidation ◽  
Maldi Mass Spectrometry ◽  
Disulfide Bridge ◽  
Covalent Modification ◽  
Oxidation Products ◽  
Affinity Tag

<p>Gliotoxin is a secondary metabolite that is produced by several species of fungi, and is toxic to mammalian cells. It is immunosuppressive, affects antigen presentation by macrophages, and causes apoptosis of some cells. Gliotoxin is an epipolythiodioxopiperazine molecule and contains an internal disulfide bridge that is highly reactive and essential for its toxicity. Suggested mechanisms of action include modification of thiol groups of cysteine residues in target proteins by generating oxidative stress or through covalent modification. The goal of this project was to develop mass spectrometry methods to detect protein modification by gliotoxin. Creatine kinase was used as a model protein. The measured mass of creatine kinase from 45 spectra gave a mean of 42,944 ± 24 which was consistent with the predicted mass of creatine kinase. A tryptic digest of creatine kinase indicated ions consistent with the predicted masses of the four cys-containing peptides including abundant ions at m/z 794, 1130 and 2870 and an ion at low intensity at 4373. The reaction of creatine kinase with gliotoxin showed a time dependent reaction that after 14 h was consistent with formation of a gliotoxin adduct. Reduction of the product with dithiothreitol released creatine kinase. Analysis of the tryptic peptides using MALDI mass spectrometry indicated complex modification of cysteines possibly including formation of a mixed disulfide adduct, intramolecular disulfides of CK, and sulfur oxidation products. Further analysis using the ICAT (isotope-coded affinity tag) method suggested modification of Cys-254 and Cys-283 by gliotoxin. Preliminary experiments examined the effects of gliotoxin on HL-60 cells using ICAT. Proteins of gliotoxin-treated and untreated cells were labeled with Heavy and Light ICAT reagents. Potential ICAT pairs were detected in the mass spectrum as a preliminary search for proteins affected by gliotoxin. The results indicate that ICAT labeling should be an effective strategy for characterization of the protein targets of gliotoxin.</p>

Abstract 231: Cloning, Expression, Charecterization and Redox Regulation of Human DDAH-1: Implications in NO Regulation

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Scott P Forbes ◽  
Lawrence Druhan ◽  
Arthur Pope ◽  
Arturo Cardounel
Keyword(s):  
Oxidative Stress ◽  
Enzyme Activity ◽  
Endothelial Dysfunction ◽  
Critical Role ◽  
Oxidation Products ◽  
Kinetic Properties ◽  
No Production ◽  
Lipid Peroxidation Product ◽  
Lipid Oxidation Products ◽  
Nos Inhibitors

Altered NO biosynthesis is known to play a central role in the pathogenesis of endothelial dysfunction. It is hypothesized that reduced NO bioavailability may result from an increase in endogenous NOS inhibitors, ADMA and NMMA, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). DDAH hydrolyzes side-chain methylated L-arg and thus regulates the activity of NOS. To date, the few studies published on DDAH have been carried out using enzyme purified from pseudomonas or rat kidney homogenates. Herein we report the cloning, expression and kinetic properties of hDDAH-1 and its regulation by redox environment. Human DDAH-1 was cloned into an e. coli expression vector and recombinant hDDAH-1 purifed to > 95% purity. Kinetic studies from the enzyme demonstrated Km values of 18.2 and 27.1 μM and Vmax values of 303 and 182 nmols/mg/min for ADMA and L-NMMA, respectively. Oxidant exposure studies were performed to determine the effects of reactive oxygen and reactive nitrogen species on DDAH activity. Results demonstrated that low level oxidant exposure had little effect on enzyme activity and that concentrations approaching 1 mM were needed to confer significant enzyme inhibition. However, exposure of hDDAH-1 to lipid oxidation products, such as 4-HNE, dose dependently inhibited DDAH activity with 15% inhibition observed at 10 μM, 50% inhibition at 50 μM and near complete inhibition at 100 μM. These levels represent pathophysiological concentrations of this lipid peroxidation product and suggest that DDAH activity can be impaired under conditions of increased oxidative stress. Proteomic analysis revealed that exposure of hDDAH-1 to 4-HNE results in Schiff base adduct formation of critical amino acid residues located within the enzyme active site. Thus, 4-HNE directly modifies hDDAH-1 with subsequent loss of enzyme activity. To determine whether this loss of DDAH activity results in NOS impairment, studies were performed using cultured endothelial cells. Results demonstrated that exposure of cells to 4-HNE (50 μM) inhibited eNOS derived NO production by 45% and reduced cellular DDAH activity by 41%. These results demonstrate a critical role for DDAH in the pathogenesis of endothelial dysfunction under conditions of oxidative stress.

scholarly journals Biomarkers of Oxidative Stress in Metabolic Syndrome and Associated Diseases

2019 ◽  
Vol 2019 ◽  
pp. 1-19 ◽  
Author(s):  
Rosa Vona ◽  
Lucrezia Gambardella ◽  
Camilla Cittadini ◽  
Elisabetta Straface ◽  
Donatella Pietraforte
Keyword(s):  
Oxidative Stress ◽  
Metabolic Syndrome ◽  
Natural Antioxidants ◽  
Therapeutic Strategies ◽  
Oxidation Products ◽  
Antioxidant Defenses ◽  
Antioxidant Systems ◽  
Associated Diseases ◽  
Lipid Oxidation Products

Metabolic syndrome (MS) represents worldwide public health issue characterized by a set of cardiovascular risk factors including obesity, diabetes, dyslipidemia, hypertension, and impaired glucose tolerance. The link between the MS and the associated diseases is represented by oxidative stress (OS) and by the intracellular redox imbalance, both caused by the persistence of chronic inflammatory conditions that characterize MS. The increase in oxidizing species formation in MS has been accepted as a major underlying mechanism for mitochondrial dysfunction, accumulation of protein and lipid oxidation products, and impairment of the antioxidant systems. These oxidative modifications are recognized as relevant OS biomarkers potentially able to (i) clarify the role of reactive oxygen and nitrogen species in the etiology of the MS, (ii) contribute to the diagnosis/evaluation of the disease’s severity, and (iii) evaluate the utility of possible therapeutic strategies based on natural antioxidants. The antioxidant therapies indeed could be able to (i) counteract systemic as well as mitochondrial-derived OS, (ii) enhance the endogenous antioxidant defenses, (iii) alleviate MS symptoms, and (iv) prevent the complications linked to MS-derived cardiovascular diseases. The focus of this review is to summarize the current knowledge about the role of OS in the development of metabolic alterations characterizing MS, with particular regard to the occurrence of OS-correlated biomarkers, as well as to the use of therapeutic strategies based on natural antioxidants.

scholarly journals Oxidative Stress and Cell Membranes in the Pathogenesis of Alzheimer's Disease

Physiology ◽  
2011 ◽  
Vol 26 (1) ◽  
pp. 54-69 ◽  
Author(s):  
Paul H. Axelsen ◽  
Hiroaki Komatsu ◽  
Ian V. J. Murray
Keyword(s):  
Oxidative Stress ◽  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Lipid Membranes ◽  
Amyloid Β ◽  
Oxidation Products ◽  
Lipid Oxidation Products ◽  
Cellular Components ◽  
The Brain ◽  
Histopathological Lesion

Amyloid β proteins and oxidative stress are believed to have central roles in the development of Alzheimer's disease. Lipid membranes are among the most vulnerable cellular components to oxidative stress, and membranes in susceptible regions of the brain are compositionally distinct from those in other tissues. This review considers the evidence that membranes are either a source of neurotoxic lipid oxidation products or the target of pathogenic processes involving amyloid β proteins that cause permeability changes or ion channel formation. Progress toward a comprehensive theory of Alzheimer's disease pathogenesis is discussed in which lipid membranes assume both roles and promote the conversion of monomeric amyloid β proteins into fibrils, the pathognomonic histopathological lesion of the disease.

scholarly journals PSIV-5 Can dietary oxidized protein induce oxidative stress in pigs?

2019 ◽  
Vol 97 (Supplement_2) ◽  
pp. 181-181
Author(s):  
Carl A Frame ◽  
Logan R Kilburn ◽  
Erika M Johnson ◽  
Mariana C Rossoni Serao
Keyword(s):  
Oxidative Stress ◽  
Companion Animals ◽  
Thiobarbituric Acid ◽  
Oxidation Products ◽  
Protein Carbonyls ◽  
P Value ◽  
Thiobarbituric Acid Reactive Substance ◽  
Oxidation Treatment ◽  
Crypt Depth ◽  
Lipid Oxidation Products

Abstract Endogenous protein oxidation as a result of oxidative stress is known to reduce the efficiency of livestock species (Boler et al., 2012; DeRouchey et al., 2004; Dibner, Atwell, Kitchell, Shermer, & Ivey, 1996). Additionally, rendered by-products are common feedstuffs in livestock diets. During processing, these sources have the potential to become oxidized. While most research on oxidative stress has focused on consumption of dietary oxidized lipids, little research has been done in the area of dietary oxidized proteins and the potential to induce oxidative stress. The objective of this study was to determine the effects of dietary oxidized protein on oxidative stress in pigs. For this study, 30 pigs 6 weeks old were divided into three dietary treatments of control, medium, and high dietary oxidized protein. Each treatment was fed the same diet, with the exception of the degree of oxidation in bovine plasma which was included in the diet at 10 percent. Pigs were fed for 19 days and then euthanized for tissues collected. Jejunum, liver, and colon were collected along with urine and plasma samples on day 0 and 18. Jejunum samples were also collected for histology. Markers of oxidative stress included protein carbonyls, thiobarbituric acid reactive substance (TBARS), 8-hydroxyguanine, and glutathione peroxidase activity. Pigs in the high oxidation treatment had an increase in crypt depth of 16 percent (p-value less than 0.05) when compared to control further resulting in an 11 percent decrease in villi height to crypt depth ratio (p-value less than 0.05). Additionally, lipid oxidation products, measured by TBARS, was 28 percent greater in the liver of pigs in the medium oxidation treatment (p-value less than 0.05) when compared to control. Even with the short duration of this study, dietary oxidized protein did impact the oxidative status of the animal. Using pigs as a model for companion animals, it could be hypothesized then that long-term exposure could have implications on longevity.

Differential PDGF secretion by graft and aortic SMC in response to oxidized LDL

2002 ◽  
Vol 283 (2) ◽  
pp. H725-H732 ◽  
Author(s):  
Afaf Absood ◽  
Akira Furutani ◽  
Tsutomu Kawamura ◽  
Linda M. Graham
Keyword(s):  
Oxidative Stress ◽  
Lipid Oxidation ◽  
Oxidized Ldl ◽  
Low Density Lipoprotein ◽  
Vascular Grafts ◽  
Density Lipoprotein ◽  
Oxidation Products ◽  
Enzyme Linked Immunosorbent Assay ◽  
Oxidized Low Density Lipoprotein ◽  
Lipid Oxidation Products

Smooth muscle cells (SMC) from prosthetic vascular grafts constitutively secrete higher levels of platelet-derived growth factor-AA (PDGF-AA) than aortic SMC. Lipid oxidation products accumulate in grafts and may stimulate PDGF production. The effect of oxidized low-density lipoprotein (oxLDL) on PDGF-AA secretion by aortic and graft SMC was compared. SMC isolated from canine thoracic aorta or Dacron thoracoabdominal grafts ( n = 10) were incubated with native LDL or oxLDL (0–400 μg/ml) for 72 h. PDGF-AA in the conditioned medium was measured with enzyme-linked immunosorbent assay. OxLDL increased PDGF-AA production by graft SMC from 78 ± 2 to 256 ± 16 pg PDGF/μg DNA and aortic SMC from 21 ± 1 to 40 ± 2 pg PDGF/μg DNA. Native LDL had no effect. N-acetylcysteine inhibited oxLDL-induced PDGF increase. Both superoxide and H2O2 stimulated PDGF secretion by graft SMC had little effect on aortic SMC. Our results suggest that PDGF production by graft (synthetic) SMC is more sensitive to stimulation by oxidative stress than aortic (contractile) SMC. Lipid oxidation products that accumulate in prosthetic vascular grafts can cause an oxidative stress, which stimulates PDGF production by graft SMC. PDGF can induce migration of aortic SMC onto the graft, contributing to the development of intimal hyperplasia.

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