Redox proteomics: identification of oxidatively modified proteins

Reactive oxygen and nitrogen species may cause various types of chemical modifications on specific proteins, Such modifications if irreversible are often associated with permanent loss of function and may lead to the elimination or to the accumulation of the damaged proteins. Reversible modifications, particularly at the cysteine residues, may have a dual role of protection from cysteine irreversible oxidation and modulation of protein function (redox regulation). Here we will review the techniques available for identifying proteins based on their redox state. In particular, we will focus on protein carbonylation, tyrosine nitration and thiol-disulfide chemistry of cysteines, with special emphasis on glutathionylation, because these are the fields where the tools of proteome analysis have been applied.     

Fluorescence thiol modification assay: oxidatively modified proteins in Bacillus subtilis

Oxidatively modified thiol groups of cysteine residues are known to modulate the activity of a growing number of proteins. In this study, we developed a fluorescence-based thiol modification assay and combined it with two-dimensional gel electrophoresis and mass spectrometry to monitor the in vivo thiol state of cytoplasmic proteins. For the Gram-positive model organism Bacillus subtilis our results show that protein thiols of growing cells are mainly present in the reduced state. Only a few proteins were found to be thiol-modified, e.g. enzymes that include oxidized thiols in their catalytic cycle. To detect proteins that are particularly sensitive to oxidative stress we exposed growing B. subtilis cells to diamide, hydrogen peroxide or to the superoxide generating agent paraquat. Diamide mediated a significant increase of oxidized thiols in a variety of metabolic enzymes, whereas treatment with paraquat affected only a few proteins. Exposure to hydrogen peroxide forced the oxidation especially of proteins with active site cysteines, e.g. of cysteine-based peroxidases and glutamine amidotransferase-like proteins. Moreover, high levels of hydrogen peroxide were observed to influence the isoelectric point of proteins of this group indicating the generation of irreversibly oxidated thiols. From the overlapping set of oxidatively modified proteins, also enzymes necessary for methionine biosynthesis were identified, e.g. cobalamin-independent methionine synthase MetE. Growth experiments revealed a methionine limitation after diamide and hydrogen peroxide stress, which suggests a thiol-oxidation-dependent inactivation of MetE. Finally, evidence is presented that the antibiotic nitrofurantoin mediates the formation of oxidized thiols in B. subtilis.     

Proteomic analysis of oxidatively modified proteins in Alzheimer's disease brain: insights into neurodegeneration.

Identification of oxidized proteins in Alzheimer's disease (AD) brain is hypothesized to lead to new insights into mechanisms of neurodegeneration and synapse loss in this dementing disorder that are associated with oxidative stress. Previous studies had shown increased oxidation of proteins in AD brain, but identifying those particular proteins that were specifically oxidized using standard immunochemical methods is a daunting task when one considers how many proteins there are in brain. To address this issue, proteomics has been used to identify specifically modified proteins in AD brain. This review outlines the nature of proteomics, the proteins identified in AD brain that are specifically oxidatively modified, and provides rational consequences related to neurodegeneration and synapse loss as sequelae to loss of function, due to oxidation and consistent with the known pathological and biochemical alteration in AD brain. The use of proteomics to learn about disease mechanisms is still embryonic, but the emerging techniques of proteomics represent a promising means to elucidate mechanisms of disease at the protein level. There are limitations to proteomics, and these, too, are discussed.     

Determination of carbonyl groups in oxidatively modified proteins by reduction with tritiated sodium borohydride

Oxidatively modified proteins have been implicated in a variety of physiologic and pathologic processes. Oxidative modification typically causes inactivation of enzymes and also the introduction of carbonyl groups into amino acid side chains of the protein. We describe a method to quantify oxidatively modified proteins through reduction of these carbonyl groups with tritiated borohydride. The technique was applied to purified, oxidatively modified glutamine synthetase and to bronchoalveolar lavage fluid from dogs and from humans. Since the protein content of lung lavage fluid is low, a very sensitive method was required to measure the oxidized residues. Reduction of the carbonyl group generated during oxidation of proteins with tritiated borohydride provided excellent sensitivity. Incorporation of tritium was directly proportional to the amount of protein with a range from 10 to 1000 micrograms. Should moieties other than amino acids be labeled, they are easily removed by rapid benchtop hydrolysis of the protein followed by chromatography on Dowex 50.     

Redox proteomics identification of oxidatively modified myocardial proteins in human heart failure: implications for protein function

Increased oxidative stress in a failing heart may contribute to the pathogenesis of heart failure (HF). The aim of this study was to identify the oxidised proteins in the myocardium of HF patients and analyse the consequences of oxidation on protein function. The carbonylated proteins in left ventricular tissue from failing (n?=?14) and non-failing human hearts (n?=?13) were measured by immunoassay and identified by proteomics. HL-1 cardiomyocytes were incubated in the presence of stimuli relevant for HF in order to assess the generation of reactive oxygen species (ROS), the induction of protein carbonylation, and its consequences on protein function. The levels of carbonylated proteins were significantly higher in the HF patients than in the controls (p<0.01). We identified two proteins that mainly underwent carbonylation: M-type creatine kinase (M-CK), whose activity is impaired, and, to a lesser extent, α-cardiac actin. Exposure of cardiomyocytes to angiotensin II and norepinephrine led to ROS generation and M-CK carbonylation with loss of its enzymatic activity. Our findings indicate that protein carbonylation is increased in the myocardium during HF and that these oxidative changes may help to explain the decreased CK activity and consequent defects in energy metabolism observed in HF.     

Role of oxidatively modified LDL in atherosclerosis

Oxidative modification of LDL is accompanied by a number of compositional and structural changes, including increased electrophoretic mobility, increased density, fragmentation of apolipoprotein B, hydrolysis of phosphatidylcholine, derivatization of lysine amino groups, and generation of fluorescent adducts due to covalent binding of lipid oxidation products to apo B. In addition, oxidation of LDL has been shown to result in numerous changes in its biologic properties that could have pathogenetic importance, including accelerated uptake in macrophages, cytotoxicity, and chemotactic activity for monocytes. The present article summarizes very recent developments related to the mechanism of oxidation of LDL by cells, receptor-mediated uptake of oxidized LDL in macrophages, the mechanism of phosphatidylcholine hydrolysis during LDL oxidation, and other biologic actions of oxidized LDL including cytotoxicity, altered eicosanoid metabolism, and effects on the secretion of growth factors and chemotactic factors. In addition, this review will examine the evidence for the presence of oxidized LDL in vivo and the evidence that oxidized LDL plays a pathogenetic role in atherosclerosis.     

Apolipoprotein-lipid association in oxidatively modified HDL and LDL

We investigated the effect of Cu²⁺ catalyzed peroxidation on the status of tryptophan (Trp) in protein moieties in HDL and LDL together with its effect on apolipoprotein-lipid association. Incubation of HDL with Cu²⁺ resulted in a rapid decrease of Trp fluorescence intensity with time with a concomitant increase in Trp maximum emission wavelength (λ_max). LDL incubated with Cu²⁺ also showed a rapid decrease in Trp fluorescence intensity with time, but with no associated increase in λ_max. The status of apo HDL and apo LDL was investigated after 4 h oxidation (4h-oxHDL and 4h-oxLDL respectively). With 4h-oxHDL, the shift in λ_max was not associated with protein dissociation but rather with protein crosslinking and formation of larger HDL species. Progressive increase in λ_max was observed in 4h-oxHDL with increase in guanidine hydrochloride (GuHCl) concentration; this was not due to protein dissociation. Although oxidation of LDL did not produce an increase in λ_max, a significant increase in wavelength was observed when 4h-oxLDL was exposed to increasing concentration of GuHCl. SDS-polyacrylamide gel electrophoresis and nondenaturing gradient gel electrophoresis of the 4h-oxLDL indicated formation of smaller molecular weight protein fragments that were still associated with LDL. Ultracentrifugation of oxidized LDL in the presence and absence of GuHCl showed no dissociated protein. In summary, these data indicate the following: (a) lipid peroxidation has a direct effect on Trp residues in both HDL and LDL, (b) oxidation of HDL is associated with conformational change in apo HDL, crosslinking and formation of larger particles, (c) oxidized HDL have a more stable apolipoprotein-lipid association than native HDL, (d) oxidation of LDL is associated with changes in apo B, that by fluorescence are apparent only in presence of GuHCl and results in fragmentation of apo B without dissociation of protein or change in particle size, and (e) stability of apolipoprotein-lipid association is comparable in oxidized and native LDL.     

In vitro labeling of proteins by reductive methylation: Application to proteins involved in supramolecular structures

Actin and tropomyosin, purified from both muscle and brain, and α-actinin, purified from muscle, have been labeled in vitro by reductive methylation to specific activities of greater than 10⁵ dpm/μg protein. Actin so modified bound DNase I and polymerized identically to unmodified actin. Furthermore, the spectral properties of actin did not change after labeling. The interactions of labeled tropomyosin and α-actinin with F-actin were nearly identical to those of the unmodified proteins. These modified proteins comigrated with their unmodified counterparts in both SDS-containing polyacrylamide gels and isoelectric focusing gels. The labeled actin was quantitatively extracted from SDS-containing polyacrylamide gels (yield > 98% of radioactivity applied demonstrating that all of the radioactivity was protein bound. The reductive methylation procedure worked well at pH 8.0–8.5 in either pyrophosphate buffer or Bicine buffer using formaldehyde with [³H]-sodium borohydride as the reducing agent. The procedure could also be performed at pH 7.0 in phosphate buffer using [¹⁴C]-formaldehyde with sodium cyanoborohydride as the reducing agent. Proteins so labeled are ideal for use in quantitative experiments involving protein-protein interactions.     

Degradation of oxidatively denatured proteins in Escherichia coli

When exposed to oxidative stress, by oxygen radicals or H2O2, E. coli exhibited decreased growth, decreased protein synthesis, and dose-dependent increases in protein degradation. The quinone menadione induced proteolysis when cells were incubated in air, but was not effective when cells were incubated without oxygen. Anaerobically grown cells also exhibited significantly lower proteolytic capacity than did cells that were grown aerobically. Xanthine plus xanthine oxidase (which generate O2- and H2O2) caused a stimulation of proteolysis which was inhibitable by catalase, but not by superoxide dismutase: Indicating that H2O2 was responsible for the increased protein degradation. Indeed, H2O2 alone was effective in inducing increased intracellular proteolysis. Two-dimensional polyacrylamide gel electrophoresis of [3H]leucine labeled E. coli revealed greater than 50% decreases in the concentrations of 10-15 cell proteins following H2O2 or menadione exposure, while several other proteins were less severely affected. To test for the presence of soluble proteases, we prepared cell-free extracts of E. coli and incubated them with radio-labeled protein substrates. E. coli extracts degraded casein and globin polypeptides at rapid rates but showed little activity with native proteins such as superoxide dismutase, hemoglobin, bovine serum albumin, or catalase. When these same proteins were denatured by exposure to oxygen radicals or H2O2, however, they became excellent substrates for degradation in E. coli extracts. Studies with albumin revealed correlations greater than 0.95 between the degree of oxidative denaturation and proteolytic susceptibility. Pretreatment of E. coli with menadione or H2O2 did not increase the proteolytic capacity of cell extracts; indicating that neither protease activation, nor protease induction were required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogenous turnover of sperm and seminal vesicle proteins in the mouse revealed by dynamic metabolic labeling

Plasticity in ejaculate composition is predicted as an adaptive response to the evolutionary selective pressure of sperm competition. However, to respond rapidly to local competitive conditions requires dynamic modulation in the production of functionally relevant ejaculate proteins. Here we combine metabolic labeling of proteins with proteomics to explore the opportunity for such modulation within mammalian ejaculates. We assessed the rate at which proteins are synthesized and incorporated in the seminal vesicles of male house mice (Mus musculus domesticus), where major seminal fluid proteins with potential roles in sperm competition are produced. We compared rates of protein turnover in the seminal vesicle with those during spermatogenesis, the timing of which is well known in mice. The subjects were fed a diet containing deuterated valine ([(2)H(8)]valine) for up to 35 days, and the incorporation of dietary-labeled amino acid into seminal vesicle- or sperm-specific proteins was assessed by liquid chromatography-mass spectrometry of samples recovered from the seminal vesicle lumen and cauda epididymis, respectively. Analyses of epididymal contents were consistent with the known duration of spermatogenesis and sperm maturation in this species and in addition revealed evidence for a subset of epididymal proteins subject to rapid turnover. For seminal vesicle proteins, incorporation of the stable isotope was evident from day 2 of labeling, reaching a plateau of labeling by day 24. Hence, even in the absence of copulation, the seminal vesicle proteins and certain epididymal proteins demonstrate considerable turnover, a response that is consonant with the capacity to rapidly modulate protein production. These techniques can now be used to assess the extent of phenotypic plasticity in mammalian ejaculate production and allocation according to social and environmental cues of sperm competition.     

Redox proteomics identification of oxidatively modified proteins in Alzheimer's disease brain and in vivo and in vitro models of AD centered around Abeta(1-42)

Alzheimer's disease is a progressive neurodegenerative disease associated with loss of memory and cognition. One hallmark of AD is the accumulation of amyloid beta-peptide (Abeta), which invokes a cascade of oxidative damage to neurons that can eventually result in neuronal death. Several markers of oxidative stress have been identified in AD brain, thus providing greater understanding into potential mechanisms involved in the disease pathogenesis and progression. In the present article, we review the application of redox proteomics to the identification of oxidized proteins in AD brain and also our recent findings on amyloid beta-peptide (Abeta)-associated in vivo and in vitro models of AD. Our redox proteomics approach has made possible the identification of specifically oxidized proteins in Alzheimer's disease (AD) brain, providing for the first time evidence on how oxidative stress plays a crucial role in AD-related neurodegeneration. The information obtained has great potential to aid in determining the molecular pathogenesis in and detecting disease markers of AD, as well as identifying potential targets for drug therapy in AD. Application of redox proteomics to study cellular events, especially related to disease dysfunction, may provide an efficient tool to understand the main mechanisms involved in the pathogenesis and progression of oxidative stress-related neurodegenerative disorders.     

The nuclear proteasome and the degradation of oxidatively damaged proteins

Summary. The accumulation of oxidized proteins is known to be linked to some severe neurodegenerative diseases like Alzheimer’s, Parkinson’s and Huntington’s disease. Furthermore, the aging process is also accompanied by an ongoing aggregation of misfolded and damaged proteins. Therefore, mammalian cells have developed potent degradation systems, which selectively degrade damaged and misfolded proteins. The proteasomal system is largely responsible for the removal of oxidatively damaged proteins form the cellular environment. Not only cytosolic proteins are prone to oxidative stress, also nuclear proteins are readily oxidized. The nuclear proteasomal system is responsible for the degradation of these proteins. This review is focused on the specific degradation of oxidized nuclear proteins, the role of the proteasome in this process and the regulation of the nuclear proteasomal system under oxidative conditions.     

Differences in the metabolism of oxidatively modified low density lipoprotein and acetylated low density lipoprotein by human endothelial cells: inhibition of cholesterol esterification by oxidatively modified low density lipoprotein

The rate of degradation of oxidatively modified low density lipoprotein (Ox-LDL) by human endothelial cells was similar to that of unmodified low density lipoprotein (LDL), and was approximately 2-fold greater than the rate of degradation of acetylated LDL (Ac-LDL). While LDL and Ac-LDL both stimulated cholesterol esterification in endothelial cells, Ox-LDL inhibited cholesterol esterification by 34%, demonstrating a dissociation between the degradation of Ox-LDL and its ability to stimulate cholesterol esterification. Further, while LDL and Ac-LDL resulted in a 5- and 15-fold increase in cholesteryl ester accumulation, respectively, Ox-LDL caused only a 1.3-fold increase in cholesteryl ester mass. These differences could be accounted for, in part, by the reduced cholesteryl ester content of Ox-LDL. However, when endothelial cells were incubated with Ac-LDL in the presence and absence of Ox-LDL, Ox-LDL led to a dose-dependent inhibition of cholesterol esterification without affecting the degradation of Ac-LDL. This inhibitory effect of Ox-LDL on cholesteryl ester synthesis was also manifest in normal human skin fibroblasts incubated with LDL and in LDL-receptor-negative fibroblasts incubated with unesterified cholesterol to stimulate cholesterol esterification. Further, the lipid extract from Ox-LDL inhibited cholesterol esterification in LDL-receptor negative fibroblasts. These findings suggest that the inhibition of cholesterol esterification by oxidized LDL is independent of the LDL and scavenger receptors and may be a result of translocation of a lipid component of oxidatively modified LDL across the cell membrane.