Malondialdehyde-modified Proteins Research Articles

The effects of short-term environment-induced heat stress on the myocardium in male mice

Environment-induced heat stress (EIHS) is caused by a sustained elevation in body temperature due to prolonged exposure to excess heat and humidity. Given the current and increasing frequency of EIHS and the large gap in knowledge regarding EIHS-mediated impacts to the myocardium, the purpose of this investigation was to determine the extent EIHS alters cardiac health. We hypothesized EIHS would cause myocardial injury and cellular dysfunction. To test this hypothesis, 13 w old male C57 mice were exposed to EIHS for 4 h or 8 h (n=7; 37.7 ± 0.10 °C, 38.3 ± 0.5 % humidity) or thermoneutral (TN) (n=7, 27.7 ± 0.15 °C, 42.5 ± 0.43% humidity) conditions, hearts were removed, weighed, and portions of the heart were fixed for histology or frozen for biochemical measures. Environment-induced HS increased subcutaneous temperature following 4 and 8 h by 0.9 °C (p< 0.01) compared to respective TN temperatures and decreased body weight by 6.8% (p=0.01) at 4 h and 13% (p<0.01) at 8 h compared to TN. Histological inspection revealed that EIHS increased anisokaryosis by 1.4-fold following 4 h (p<0.01) and by 75% following 8 h (p=0.01) compared to TN animals. Environment-induced HS increased relative abundance of malondialdehyde-modified proteins (35%, p=0.02), a marker of oxidative stress, and increased relative protein abundance of SOD2 (72%, p=0.05) following 4 h compared to TN. Eight hours of EIHS increased relative protein abundance of SOD1 (69%, p=0.01) compared to TN and increased catalase and GPX1 compared to TN (91%, p<0.01; 2-fold, p<0.01) and compared to 4 h of EIHS (72%, p=0.03; 34%, p=0.01), though MDA-modified proteins were similar to TN. Following 4 h of EIHS, markers of mitochondrial abundance, including electron transport chain complexes II (69%, p=0.02) and V (36%, p=0.03), VDAC2 (79%, p=0.01) and PHB1 (65%, p=0.03) were increased compared to TN, and PDH (35%, p=0.04) and Cox IV (55%, p=0.01) were increased compared to 8 hrs of EIHS. Environment-induced HS appeared to alter mitochondrial fusion and fission as DRP1 (51%, p=0.05), phosphorylated (p)-DRP1 (47%, p=0.04), FIS1 (57%, p=0.01) and Mfn1 (26%, p=0.03) were increased compared to TN following 4 h and Mfn2 was increased (37%, p=0.02) following 8 h of EIHS compared to TN. Mitophagy markers BNIP3L/NIX and Pink1 were increased by EIHS following 4 h compared to TN (78%, p=0.02; 41%, p=0.03). Environment-induced HS increased autophagy markers ULK1 (36%, p<0.01), PI3K (80%, p<0.01), and ATG7 (49%, p<0.01) following 4 h compared to TN and ULK1 (33%, p<0.01) and ATG7 (41%, p=0.02) following 4 h compared to 8 h of EIHS. However, p-ULK1, Beclin, p-Beclin, ATG12, and ATG16 were similar between groups. Relative protein abundance of p62 and LC3 II were similar between groups; however, using a colchicine-inhibition approach, 8 h of EIHS caused a significant accumulation of p62, suggestive of increased flux. These data suggest that in a murine model of EIHS, 4 h was suffcient to cause cellular injury and dysfunction in the myocardium; however, as EIHS continued, it was associated with changes reflective of a cellular stress response that may contribute to acclimation. This work was supported by USDA 2020-02716. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A Comprehensive Analytical Strategy To Identify Malondialdehyde-Modified Proteins and Peptides.

Mass spectrometric-based proteomics is a powerful tool to analyze post-translationally modified proteins. Carbonylation modifications that result from oxidative lipid breakdown are a class of post-translational modifications that are poorly characterized with respect to protein targets and function. This is partly due to the lack of dedicated mass spectrometry-based technologies to facilitate the analysis of these modifications. Here, we present a comprehensive approach to identify malondialdehyde-modified proteins and peptides. Malondialdehyde is among the most abundant of the lipid peroxidation products; and malondialdehyde-derived adducts on proteins have been implicated in cardiovascular diseases, neurodegenerative disorders, and other clinical conditions. Our integrated approach targets three levels of the overall proteomic workflow: (i) sample preparation, by employing a targeted enrichment strategy; (ii) high-performance liquid chromatography, by using a gradient optimized for the separation of the modified peptides; and (iii) tandem mass spectrometry, by improving the spectral quality of very low-abundance peptides. By applying the optimized procedure to a whole cell lysate spiked with a low amount of malondialdehyde-modified proteins, we were able to identify up to 350 different modified peptides and localize the modification to a specific lysine residue. This methodology allows the comprehensive analysis of malondialdehyde-modified proteins.

2,5-hexanedione induced apoptosis of rat bone marrow mesenchymal stem cells by reactive oxygen species.

n-Hexane, a common industrial organic solvent, causes multiple organ damage, especially neurotoxicity, which is proved to be caused by its metabolite 2,5-hexanedione (2,5-HD). We previously showed that 2,5-HD induced apoptosis of rat bone marrow mesenchymal stem cells (BMSCs). In the current study, we explored the mechanism of 2,5-HD-induced apoptosis, especially the role played by reactive oxygen species (ROS). Intracellular ROS levels after 2,5-HD treatment were measured by the dichloro-dihydro-fluorescein diacetate (DCFH-DA) method, and the antioxidant N-acetyl cysteine (NAC) was used to scavenge ROS. Apoptosis, mitochondrial membrane potential (MMP), and caspase-3 activity were measured after 2,5-HD exposure with or without NAC pretreatment. In rat BMSCs, 20 mM 2,5-HD significantly increased ROS levels and apoptosis. In addition, MMP activity was decreased and caspase-3 activity was increased. With NAC pretreatment, ROS increases were prevented, cells were rescued from apoptosis, and both MMP and caspase-3 activity returned to normal levels. Western blotting analysis of malondialdehyde-modified proteins and superoxide dismutase (SOD) 1 showed that after 2,5-HD exposure, BMSCs had oxidative damage and abnormal SOD1 expression. These returned to normal when cells were pretreated with NAC in addition to 20 mM 2,5-HD. Furthermore, the expressions of NF-κB p65/RelA and phospho-NF-κB p65/RelA (Ser536) were suppressed after 2,5-HD exposure and restored by NAC pretreatment. 2,5-HD-induced apoptosis in rat BMSCs is potentially mediated by excessive ROS production.

Inhibition of cytochrome P450 2E1 and activation of transcription factor Nrf2 are renoprotective in myoglobinuric acute kidney injury

Rhabdomyolysis accounts for ∼10% of acute kidney injuries. In glycerol-induced myoglobinuric acute kidney injury, we found an increase in the nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear protein, a key redox-sensitive transcription factor, and Nrf2-regulated genes and proteins including upregulation of heme oxygenase-1. In in vitro studies, pretreatment of LLC-PK1 cells with an activator of Nrf2 before myoglobin exposure significantly decreased oxidant generation and cytotoxicity, whereas Nrf2 inhibition and gene silencing exacerbated the injury. Chlormethiazole, a specific CYP2E1 transcription inhibitor, prevented an increase in catalytic iron in the kidneys, decreased oxidative stress, blocked nuclear translocation of the Nrf2 protein, decreased heme oxygenase-1 upregulation, and provided functional and histological protection against acute kidney injury. CYP2E1 inhibitors and gene silencing in renal tubular epithelial cells significantly decreased reactive oxygen species generation and provided marked protection against myoglobin-induced cytotoxicity. Thus, during CYP2E1-induced oxidative stress, the transcription factor Nrf2 has a pivotal role in the early adaptive response. Inhibition of CYP2E1 coupled with the prior induction of Nrf2 may be a valuable tool to reduce CYP2E1-mediated rhabdomyolysis-induced acute kidney injury.

Recognition of Malondialdehyde-modified Proteins by the C Terminus of Complement Factor H Is Mediated via the Polyanion Binding Site and Impaired by Mutations Found in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a severe thrombotic microangiopathy characterized by uncontrolled complement activation against endothelial and blood cells. Mutations in the C-terminal target recognition domains 19-20 of complement regulator factor H (FH) are strongly associated with aHUS, but the mechanisms triggering disease onset have remained unresolved. Here we report that several aHUS-related mutations alter the binding of FH19-20 to proteins where lysines have reacted with malondialdehyde (MDA). Although FH19-20 did not interact with MDA-modified hexylamine, lysine-containing peptides, or a proteolytically degraded protein, it bound to MDA-modified polylysine. This suggests that FH19-20 recognizes only clustered MDA adducts. Binding of MDA-modified BSA to FH19-20 was ionic by nature, depended on positive residues of FH19-20, and competed with the polyanions heparin and DNA. This could not be explained with the mainly neutral adducts known to form in MDA modification. When positive charges of lysines were eliminated by acetic anhydride instead of MDA, the acetylated BSA started to bind FH19-20. Together, these results indicate that negative charges on the modified proteins dominate the interaction with FH19-20. This is beneficial for the physiological function of FH because by binding to the negative charges of the modified target, FH could prevent excess complement activation initiated by naturally occurring antibodies recognizing MDA epitopes with multiple different structures. We propose that oxidative stress leading to formation of MDA adducts is a common feature for triggers of aHUS and that failure of FH in protecting MDA-modified surfaces from complement activation is involved in the pathogenesis of the disease.

Cytochrome P450 2B1 Mediates Complement-dependent Sublytic Injury in a Model of Membranous Nephropathy

Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochrome P450 2B1 (CYP2B1) in this complement-mediated sublytic injury. Overexpression of CYP2B1 in GECs significantly increased the formation of reactive oxygen species, cytotoxicity, and collapse of the actin cytoskeleton following treatment with anti-tubular brush-border antiserum (anti-Fx1A). In contrast, silencing of CYP2B1 markedly attenuated anti-Fx1A-induced reactive oxygen species generation and cytotoxicity with preservation of the actin cytoskeleton. Gelsolin, which maintains an organized actin cytoskeleton, was significantly decreased by complement C5b-9-mediated injury but was preserved in CYP2B1-silenced cells. In rats injected with anti-Fx1A, the cytochrome P450 inhibitor cimetidine blocked an increase in catalytic iron and ROS generation, reduced the formation of malondialdehyde adducts, maintained a normal distribution of nephrin in the glomeruli, and provided significant protection at the onset of proteinuria. Thus, GEC CYP2B1 contributes to complement C5b-9-mediated injury and plays an important role in the pathogenesis of passive Heymann nephritis.

Enhanced reactivity to malondialdehyde-modified proteins by systemic lupus erythematosus autoantibodies

Objective: Evaluation of the reactivity of autoantibodies of systemic lupus erythematosus (SLE) patients directed against malondialdehyde (MDA)-modified catalase, superoxide dismutase (SOD), and different Hep2 protein fractions (hydrophobic, hydrophilic, and nuclear).Method: Thiol groups and MDA–protein adducts were first assessed among 65 SLE patients and 60 healthy controls. Then, the reactivities of SLE immunoglobulin (Ig)G autoantibodies towards MDA-modified and unmodified proteins were compared using a standard enzyme-linked immunosorbent assay (ELISA).Results: An increase in the levels of MDA-modified proteins and a decrease in the concentration of thiol groups among SLE patients (p < 0.05) were observed. IgG circulating autoantibodies in the sera of SLE patients exhibited a significant enhanced reactivity (p < 0.05) against catalase and SOD-modified proteins. The same data were observed in the different protein fractions extracted from cultured cells (p < 0.05).Conclusion: These data reinforce the role of oxidative stress and especially lipid peroxidation products in the progression of SLE disease.

Hindlimb unloading decreases thioredoxin-related antioxidant proteins and increases thioredoxin-binding protein-2 in rat skeletal muscle

To investigate role(s) of thioredoxin-related antioxidant proteins in disuse muscle atrophy, we examined the levels of thioredoxin-1 (Trx-1), peroxiredoxin-3/SP-22 (Prx-3) and thioredoxin-binding protein-2 (TBP-2) in rat soleus muscle subjected to hindlimb unloading (HU) for 2, 4, 7 or 14 days. The muscle weight loss was initially observed on day 4. The increases in aclorein- and malondialdehyde-modified proteins, and the decreases in the levels of Trx-1, Prx-3 and Mn-SOD were observed in the late phase of muscle atrophy, whereas, the increase in mRNA expression of TBP-2, a negative regulator of thioredoxin, preceded muscle atrophy. These findings suggest that the decrease of those antioxidant proteins, particularly a marked decrease of Trx-1, may be responsible for the enhanced oxidative damage during the late phase of disuse muscle atrophy. Furthermore, the increase in TBP-2 preceding the muscle atrophy may suppress the thioredoxin-mediated redox signaling, which can be an initial trigger leading to disuse muscle atrophy.

Circulating antibodies recognizing malondialdehyde-modified proteins in healthy subjects

Antibodies against malondialdehyde (MDA)-modified proteins are often increased in patients with diseases related to oxidative stress. However, the clinical significance of these antibodies is hampered by their frequent presence also in healthy controls. Aim of this work has been to characterize the immune reactivity against MDA-derived antigens in healthy subjects. The sera of 120 healthy subjects contained IgG and IgM targeting MDA-modified human albumin (HSA), fibrinogen, and LDL. These sera also displayed weak reactivity with oxidized LDL and HSA complexed with oxidized arachidonic acid. Conversely, oxidized HSA or HSA complexed with other aldehydic lipid peroxidation products was not recognized. Control sera also did not recognize cyclic dihydropyridine-MDA products, while HSA-MDA reactivity was associated ( r > 0.9; p < .0005) with the presence of fluorescent lysine-conjugated-imine cross-links. In Western blots both IgG and IgM recognized high molecular weight HSA-MDA aggregates, but not monomeric HSA-MDA adducts. The addition of sodium cyanoborohydride, that prevented conjugated-imine fluorescence and protein aggregation during HSA-MDA preparation, abolished the antibody binding. This suggested that the plasma of healthy subjects contained IgG and IgM recognizing protein aggregates linked through 1-amino-3-imino-propene bridges. The function of these antibodies is at the moment unknown, but they might contribute to scavenging MDA cross-linked proteins.

Age-related changes in the thyroid hormone effects on malondialdehyde-modified proteins in the rat heart.

To determine the age-related changes in thyroid hormone (TH) effects on malondialdehyde (MDA)-modified proteins in cardiac tissue, rats at 4, 12, and 25 months of age were studied. Hyperthyroidism was induced with daily injection of L-triiodothyronine (15 microg/100 g) intraperitoneally for 10 days. Hypothyroidism was induced with 0. 025% methimazole in the drinking water for 4 weeks. MDA proteins were measured with immunoblots using a specific anti-MDA antiserum. MDA was measured as thiobarbituric acid reactive substance. Hypothyroidism in 4-month-old rats was associated with significant reduction in MDA proteins compared to euthyroid rats (13.4 +/- 5.9% vs. 99.8 +/- 10.4% of controls P < 0.001). Hyperthyroidism did not result in a significant change of MDA proteins. In aged rats, neither hypothyroidism nor hyperthyroidism was associated with significant changes in cardiac MDA proteins. The changes in MDA proteins did not correlate with cardiac MDA concentrations. In young rats, the MDA concentrations (nmol/mg) were significantly reduced in hypothyroidism (2.71 +/- 0.21) and were increased in hyperthyroidism (8.19 +/- 0.78) compared to euthyroid values (5.06 +/- 0.71) P < 0. 01. In aged rats, cardiac MDA content was significantly increased during both hyperthyroidism and hypothyroidism. We conclude that alterations in MDA protein content is yet another potential biochemical effect of TH in cardiac tissue. This particular effect is significantly blunted with age.

Antibodies against malondialdehyde-modified proteins. Induction and ELISA measurement of specific antibodies.

AbstractPolyunsaturated fatty acids undergo in vivo oxidative damage called lipid peroxidation, breaking down to lipid hydroperoxides and various secondary products (1). Increase in lipid peroxides have been reported, for example, in radiation damage, diabetes, hyperlipidemia, and vascular diseases, and there is considerable interest in the relation between oxidative stress and development of atherosclerosis (2,3). KeywordsWitness PatientCitric Acid MonohydrateRabbit ImmunizationAcidic Phosphate BufferRabbit Serum AlbuminThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Diminished Susceptibility to Proteolysis after Protein Modification by the Lipid Peroxidation Product Malondialdehyde: Inhibitory Role for Crosslinked and Noncrosslinked Adducted Proteins

The lipid peroxidation product malondialdehyde forms adducts with proteins that are detected during routine assays for protein carbonylation. To test whether this damage alters the susceptibility of a protein to proteolysis, we treated bovine serum albumin with various concentrations of malondialdehyde and examined its susceptibility to digestion by α-chymotrypsin. In keeping with findings concerning the consequences of protein damage by other carbonyl products of lipid peroxidation, we found that malondialdehyde-modified protein was resistant to proteolysis. Since significant protein crosslinking occurred during modification with malondialdehyde, we investigated the possibility that crosslinked proteins were acting as proteolytic inhibitors. Malondialdehyde-modified proteins were resolved into crosslinked and noncrosslinked forms and the effectiveness of both species as proteolytic antagonists was examined. While both forms of malondialdehyde-adducted proteins were more potent proteolytic inhibitors than unmodified albumin, there were no significant differences in inhibitory potency between crosslinked and noncrosslinked proteins. Our findings suggest that malondialdehyde-modification produces protease-resistant proteins without an obligatory role for crosslinking.

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Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. A recent immunohistochemical study has revealed that the materials immunoreactive to the antibody against the aldehyde (4-hydroxy-2-nonenal)-modified proteins are generated in the renal proximal tubules of rats treated with Fe-NTA (S. Toyokuni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620). Here we present further evidences that cytosolic proteins modified with lipid peroxidation-derived aldehydes (4-hydroxy-2-nonenal and malondialdehyde) are indeed accumulated in the kidney of rats treated with Fe-NTA. A single intraperitoneaI Fe-NTA treatment (15 or 30 mg Fe/kg body wt) induced a rapid accumulation of thiobarbituric acid-reactive substances, a direct consequence of membrane lipid peroxidation, mostly within the cytosolic compartment of rat kidney. The accumulation of lipid peroxidation-derived aldehydes was associated with an apparent accumulation (from 67.9 nmol in untreated controls to 83.3 nmol/mg protein in Fe-NTA-treated rats at 3 h after treatment) and subsequent elimination of aberrant proteins as assessed by a 2,4-dinitrophenylhydrazine postlabeling assay. Immunoblot analysis with two different polyclonal antibodies against 4-hydroxy-2-nonenal-modified and malondialdehyde-modified proteins detected a temporary accumulation of aldehyde-modified proteins in the cytosol. An immunohistochemical technique with serial sectioning revealed that immunoreactivities of these antibodies were observed in the identical portion of the renal proximal tubules. The implications of these data in relation to the nephrotoxicity of Fe-NTA-are discussed.

A Renal Carcinogen Ferric Nitrilotriacetate Mediates a Temporary Accumulation of Aldehyde-Modified Proteins Within Cytosolic Compartment of Rat-Kidney

Iron overload with ferric nitrilotriacetate (Fe-NTA) induces acute renal proximal tubular necrosis, a consequence of oxidative tissue damage, that eventually leads to a high incidence of renal adenocarcinoma in rodents. A recent immunohistochemical study has revealed that the materials immunoreactive to the antibody against the aldehyde (4-hydroxy-2-nonenal)-modified proteins are generated in the renal proximal tubules of rats treated with Fe-NTA (S. Toyokuni et al. (1994) Proc. Natl. Acad. Sci. USA 91, 2616-2620). Here we present further evidences that cytosolic proteins modified with lipid peroxidation-derived aldehydes (4-hydroxy-2-nonenal and malondialdehyde) are indeed accumulated in the kidney of rats treated with Fe-NTA. A single intraperitoneaI Fe-NTA treatment (15 or 30 mg Fe/kg body wt) induced a rapid accumulation of thiobarbituric acid-reactive substances, a direct consequence of membrane lipid peroxidation, mostly within the cytosolic compartment of rat kidney. The accumulation of lipid peroxidation-derived aldehydes was associated with an apparent accumulation (from 67.9 nmol in untreated controls to 83.3 nmol/mg protein in Fe-NTA-treated rats at 3 h after treatment) and subsequent elimination of aberrant proteins as assessed by a 2,4-dinitrophenylhydrazine postlabeling assay. Immunoblot analysis with two different polyclonal antibodies against 4-hydroxy-2-nonenal-modified and malondialdehyde-modified proteins detected a temporary accumulation of aldehyde-modified proteins in the cytosol. An immunohistochemical technique with serial sectioning revealed that immunoreactivities of these antibodies were observed in the identical portion of the renal proximal tubules. The implications of these data in relation to the nephrotoxicity of Fe-NTA-are discussed.

Age-related changes in tissue content of malondialdehyde-modified proteins

One of the possible mechanisms of the age-related modifications of proteins is the result of reaction with malondialdehyde (MDA), a lipid peroxidation byproduct. To determine the effect of age on the extent of MDA derivatization of proteins in plasma and various tissues, male Fisher 344 rats at 4,12 and 26 months of age were studied. Protein electrophoresis and immunoblotting was carried out using a specific antiserum against MDA-protein complexes. The concentration of MDA-proteins in plasma (mean ± SD of optical density in 50 μg protein) was 201.6 ± 47.7 in 4 month old rats, 197.4 ± 67 in 12 month old rats and 101.4 ± 22.7 in 26 month old rats (P<0.01). The MDA protein content of testicle, liver and heart was increased in 12 month old rats compared to 4 and 26 month old rats. There were no age-related differences in MDA-protein content of lung, brain, and kidney. Because of the interindividual variability of MDA-protein profiles within an age group distinct age-related changes in the distribution of various MDA protein bands could not be documented.