Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an age-related, fatal motor neuron degenerative disease occurring both sporadically (sALS) and heritably (fALS), with inherited cases accounting for approximately 10% of diagnoses. Although multiple mechanisms likely contribute to the pathogenesis of motor neuron injury in ALS, recent advances suggest that oxidative stress may play a significant role in the amplification, and possibly the initiation, of the disease. Lipid peroxidation is one of the several outcomes of oxidative stress. Since the central nervous system (CNS) is enriched with polyunsaturated fatty acids, it is particularly vulnerable to membrane-associated oxidative stress. Peroxidation of cellular membrane lipids or circulating lipoprotein molecules generates highly reactive aldehydes, among which is 4-hydroxy-2-nonenal (HNE). HNE levels are increased in spinal cord motor neurons of ALS patients, indicating that lipid peroxidation is associated with the motor neuron degeneration in ALS. In the present study, we used a parallel proteomic approach to identify HNE-modified proteins in the spinal cord tissue of a model of fALS, G93A-SOD1 transgenic mice, in comparison to the nontransgenic mice. We found three significantly HNE-modified proteins in the spinal cord of G93A-SOD1 transgenic mice: dihydropyrimidinase-related protein 2 (DRP-2), heat-shock protein 70 (Hsp70), and possibly alpha-enolase. These results support the role of oxidative stress as a major mechanism in the pathogenesis of ALS. Structural alteration and activity decline of functional proteins may consistently contribute to the neurodegeneration process in ALS.     

A method for detection of 4-hydroxy-2-nonenal adducts in proteins

We developed a procedure to measure 4-hydroxy-2-nonenal (HNE)-amino acid adducts using the fluorescent probe 2-aminopyridine (2-AP). The method is based on the fact that HNE forms Michael addition-type amino acid adducts possessing an aldehyde functionality, which upon reaction with 2-AP in the presence of NaBH₃CN can be converted to their pyridylaminated derivatives. The HNE-amino acid adducts, namely Michael addition-type HNE-cysteine, HNE-histidine, and HNE-lysine adducts, after pyridylamination were resistant to conventional acid-hydrolysis conditions for protein (6 N HCl/110 °C/24 h) and could be detected by HPLC with a fluorescence detector. The reductive amination-based fluorescent labeling of HNE adducts is a simple and accurate technique that may be widely used to reveal increased levels of covalently modified proteins with HNE and its related aldehydes during aging and disease.     

Compartmentalization and regulation of iron metabolism proteins protect male germ cells from iron overload

The universal importance of iron, its high toxicity, and complex chemistry present a challenge to biological systems in general and to protected compartments in particular. The high mitotic rate and avid mitochondriogenesis of developing male germ cells imply high iron requirements. Yet access to germ cells is tightly regulated by the blood-testis barrier that protects the meiotic and postmeiotic germ cells. To elucidate how iron is supplied to developing male germ cells, we analyzed iron deposition and iron transport proteins in testes of mice with iron overload and with genetic ablation of the iron regulators Hfe and iron regulatory protein 2. Iron accumulated mainly around seminiferous tubules, and only small amounts localized within the seminiferous tubules. The localization and regulation of proteins involved in iron import, storage, and export such as transferrin, transferrin receptor, the divalent metal transporter-1, cytosolic ferritin, and ferroportin strongly support a model of a largely autonomous iron cycle within seminiferous tubules. We show evidence that ferritin secretion from Sertoli cells may play an important role in iron acquisition of primary spermatocytes. During spermatogenic development iron is carried along from primary spermatocytes to spermatids, and from spermatids iron is recycled to the apical compartment of Sertoli cells, which traffic it back to a new generation of spermatocytes. Losses are replenished by the peripheral circulation. Such an internal iron cycle essentially detaches the iron homeostasis within the seminiferous tubule from the periphery and protects developing germ cells from iron fluctuations. This model explains how compartmentalization can optimize cellular and systemic nutrient homeostasis.     

Modifications of proteins by 4-hydroxy-2-nonenal in the ventilatory muscles of rats

Although 4-hydroxy-2-nonenal (HNE, a product of lipid peroxidation) is a major cause of oxidative damage inside skeletal muscles, the exact proteins modified by HNE are unknown. We used two-dimensional electrophoresis, immunoblotting, and mass spectrometry to identify selective proteins targeted by HNE inside the diaphragm of rats under two conditions: severe sepsis [induced by E. coli lipopolysaccharides (LPS)] and during strenuous muscle contractions elicited by severe inspiratory resistive loading (IRL). Diaphragm HNE-protein adduct formation (detected with a polyclonal antibody) increased significantly after 1 and 3 h of LPS injection with a return to baseline values thereafter. Similarly, HNE-protein adduct formation inside the diaphragm rose significantly after 6 but not 3 h of IRL. Mass spectrometry analysis of HNE-modified proteins revealed enolase 3b, aldolase and triosephosphate isomerase 1, creatine kinase, carbonic anyhdrase III, aconitase 2, dihydrolipoamide dehydrogenase, and electron transfer flavoprotein-beta. Measurements of in vitro enolase activity in the presence of pure HNE revealed that HNE significantly attenuated enolase activity in a dose-dependent fashion, suggesting that HNE-derived modifications have inhibitory effects on enzyme activity. We conclude that lipid peroxidation products may inhibit muscle contractile performance through selective targeting of enzymes involved in glycolysis, energy production as well as CO(2) hydration.     

Hepatic distribution of iron, copper, zinc and cadmium-containing proteins in normal and iron overload mice

Subcellular distribution of metal-containing proteins of Fe, Cu, Zn and Cd were determined in the liver samples of iron overload mice by size exclusion high performance liquid chromatography with on-line coupling to UV and inductively coupled plasma mass spectrometry. Collision cell techniques was used to remove polyatomic interferences for some elements, such as Fe. Comparative molecular weight (MW) information of the elemental fraction was obtained within a retention time of 40 min. Fe was present only in high-MW (HMW) protein; Cu, Zn and Cd were found in different MW proteins. It was also observed that these four elements studied showed predominant association with HMW fractions. Moreover, compared with the normal group, we found that the contents of these elements except Cu significantly increased and the distribution of some elements like Cd changed in iron overload mouse liver. It means that excessive iron accumulation in vivo may affect the metabolism of other element such as Zn and Cd.     

Heat shock preconditioning reduces the formation of 8-hydroxy-2'-deoxyguanosine and 4-hydroxy-2-nonenal modified proteins in ischemia-reperfused liver of rats

Heat shock preconditioning (HSPC) is a promising strategy for providing ischemic tolerance. The objective of this study is to investigate the effectiveness of HSPC in preventing oxidative damage of cellular proteins and DNA during ischemia-reperfusion of the liver. Male Wistar rats were divided into a heat shock group (group HS) and control (group C). Forty-eight hours prior to ischemia, rats in group HS received HSPC at 42 degrees C for 15 min. All rats received hepatic warm ischemia for 30min and subsequent reperfusion. The formation of 8-hydroxy-2'-deoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (HNE) modified proteins in liver tissue, survival rate of the animals, and changes in biochemical and histological parameters were compared between groups. Heat shock protein 72 was produced only in group HS. The 7-day survival of rats was significantly better in group HS (10/10) than in group C (5/10) (p < 0.01). The serum release of alanine aminotransferase (n = 10, p < 0.01) and the concentration of adenosine triphosphate in liver tissue (n = 10, p < 0.01) 40min after reperfusion was significantly better in group HS than in group C. The formation of 8-OHdG in liver tissue measured by high-performance liquid chromatography was suppressed in group HS (p < 0.01). The production of HNE-modified proteins as determined by Western-blot analysis was also decreased in group HS. These results were also confirmed by immunohistochemical analysis. As determined by levels of 8-OHdG and HNE-modified proteins produced during ischemia-reperfusion of the liver, HSPC reduced the oxidative injury of cellular proteins and DNA in the liver tissue.     

Serum ferritin in iron overload

Even with uncomplicated iron overload, serum ferritin which can be identified in the circulating blood by sensitive immunochemical methods has a direct and quantitative correlation to the iron stored in the organism. The relation of stored iron and serum ferritin is not linear, but has an exponential character. The diagnostic function of serum ferritin as an indicator of stored iron, however, is virtually not influenced by it. The indications listed in Tab. 3 can be demarcated for diagnostic application in cases of iron overload. Hitherto, the molecular microheterogenicity of serum ferritin has exercised no essential impact on its diagnostic application. High ferritin concentrations may arise in the circulating blood by a number of disease processes listed in Tab. 4, without the simultaneous existence of a respective iron overload of the tissue. These correlations have to be observed in the diagnostic application of determining serum ferritin as well as in methodical possibilities of fault (high dose hook effect), thus limiting the use of serum ferritin as an indicator of stored iron both in case of iron overload and iron deficiency. As in all isolated laboratory investigations, all other clinical and chemical laboratory information available about the individual patient has to be taken into account in each case for interpreting the serum ferritin concentration.     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

4-Hydroxy-2-nonenal-modified glyceraldehyde-3-phosphate dehydrogenase is degraded by cathepsin G

Degradation of oxidized or oxidatively modified proteins is an essential part of the antioxidant defenses of cells. 4-Hydroxy-2-nonenal (HNE), a major reactive aldehyde formed by lipid peroxidation, causes many types of cellular damage. It has been reported that HNE-modified proteins are degraded by the ubiquitin–proteasome pathway or, in some cases, by the lysosomal pathway. However, our previous studies using U937 cells showed that HNE-modified glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is degraded by an enzyme that is sensitive to a serine protease inhibitor, diisopropyl fluorophosphate (DFP), but not a proteasome inhibitor, MG-132, and that its degradation is not catalyzed in the acidic pH range where lysosomal enzymes are active. In the present study, we purified an HNE-modified GAPDH-degrading enzyme from a U937 cell extract to a final active fraction containing two proteins of 28 kDa (P28) and 27 kDa (P27) that became labeled with [³H]DFP. Using peptide mass fingerprinting and a specific antibody, P28 and P27 were both identified as cathepsin G. The degradation activity was inhibited by cathepsin G inhibitors. Furthermore, a cell extract from U937 cells transfected with a cathepsin G-specific siRNA hardly degraded HNE-modified GAPDH. These results suggest that cathepsin G plays a role in the degradation of HNE-modified GAPDH.     

Localization of bismuth radiotracer in rat kidney following exposure to bismuth

It has been proposed that alpha emitting 212Bi (t1/2 = 60 min) coupled to tumor-specific antibodies may be a useful radiotherapeutic agent. However, since Bi can accumulate in the kidney, it is necessary to characterize the factors influencing localization of Bi within this tissue in order to evaluate the potential for radiation damage to the renal system. In this study, the localization of Bi radiotracers was determined in kidneys of rats previously exposed and not exposed to mumole quantities of Bi. Following repeated injection of Bi (4 x 14 mumols (3 mg Bi)/kg bw) the element accumulated mainly in the kidney followed by liver, spleen, pancreas, bone, and brain. Kidney copper and liver zinc concentrations were higher in Bi-exposed rats than in non-exposed rats. Within the cytosol, in Bi-exposed rats, Bi radiotracer in the kidney was associated with a metallothionein-like protein (Mt). In contrast, non-exposed rats contained no detectable metallothionein-like proteins in the kidney and the Bi tracer was associated with the hemoglobin fraction of the cell. Thus, when Bi is administered in tracer quantities such as that incorporated for use as a radiopharmaceutical, no induction of, and association with, metallothionein-like proteins should occur. These results suggest that the potential nephrotic effects of 212Bi will be influenced by the individual's previous exposure to Bi-containing drugs, or other metallothionein-inducing insults.     

Altered expression of iron transport proteins in streptozotocin-induced diabetic rat kidney

Diabetes mellitus is associated with altered iron homeostasis in both human and animal diabetic models. Iron is a metal oxidant capable of generating reactive oxygen species (ROS) and has been postulated to contribute to diabetic nephropathy. Two proteins involved in iron metabolism that are expressed in the kidney are the divalent metal transporter, DMT1 (Slc11a2), and the Transferrin Receptor (TfR). Thus, we investigated whether renal DMT1 or TfR expression is altered in diabetes, as this could potentially affect ROS generation and contribute to diabetic nephropathy. Rats were rendered diabetic with streptozotocin (STZ-diabetes) and renal DMT1 and TfR expression studied using semi-quantitative immunoblotting and immunofluorescence. In STZ-diabetic Sprague-Dawley rats, renal DMT1 expression was significantly reduced and TfR expression increased after 2 weeks. DMT1 downregulation was observed in both proximal tubules and collecting ducts. Renal DMT1 expression was also decreased in Wistar rats following 12 weeks of STZ-diabetes, an effect that was fully corrected by insulin-replacement but not by cotreatment with the aldose reductase inhibitor, sorbinil. Increased renal TfR expression was also observed in STZ-diabetic Wistar rats together with elevated cellular iron accumulation. Together these data demonstrate renal DMT1 downregulation and TfR upregulation in STZ-diabetes. Whilst the consequence of altered DMT1 expression on renal iron handling and oxidant damage remains to be determined, the attenuation of the putative lysosomal iron exit pathway in proximal tubules could potentially explain lysosomal iron accumulation reported in human diabetes and STZ-diabetic animals.     

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.     

4-hydroxy-2-nonylquinoline: a novel iron chelator isolated from a bacterial cell membrane

The membrane associated iron chelator of Pseudomonas aeruginosa has been extracted from membranes of iron-rich cells with ethanol and purified by reverse phase HPLC. Using 13C NMR and FAB mass spectroscopy, the structure of the chelator has been determined to be 4-hydroxy-2-nonylquinoline. This compound has been previously isolated and named pseudan IX, a name which we use here. We synthesized pseudan IX and show that the spectral properties of the synthesized compound and the purified compound are nearly identical. Also purified from the ethanol extract of membranes is 4-hydroxy-2-heptylquinoline, i.e., pseudan VII. Bacterially purified pseudan IX binds iron as indicated by the incorporation of radiolabeled iron into the chelator and by the formation of pink micelles in a concentrated ethanol extract. The formation of pink micelles upon addition of iron to the synthesized compound indicates that it binds iron.     

Post-transfusional iron overload in the haemoglobinopathies

In this report, we review the recent advances in evaluation and treatment of transfusional iron overload (IO). Results of the French thalassaemia registry are described. According to the disease, thalassaemia major or sickle cell anaemia, mechanisms and toxicity of iron overload, knowledge about IO long-term outcome and chelation treatment results, respective value of IO markers, differ. The recent tools evaluating organ specific IO and the diversification of iron chelator agents make possible to individualize chelation therapy in clinical practice. The severity of IO and the level of transfusional iron intake, the preferential localization of IO (heart/liver) as well as the tolerance and adherence profiles of the patient can now be taken into account. Introduction of cardiac magnetic resonance imaging for the quantification of myocardial iron and use of oral chelators have already been reported as decreasing the cardiac mortality rate related to IO in thalassaemia major patients. Long-term observation of patients under oral chelators will show if morbidity is also improving via a more continuous control of toxic iron and/or a better accessibility to cellular iron pools.     

Copper overload affects copper and iron metabolism in Hep-G2 cells

Divalent metal transporter #1 (DMT1) is responsible for intestinal nonheme Fe apical uptake. However, DMT1 appears to have an additional function in Cu transport in intestinal cells. Because the liver has an essential role in body Cu homeostasis, we examined the potential involvement of Cu in the regulation of DMT1 expression and activity in Hep-G2 cells. Cells exposed to 10 microM Cu exhibited a 22-fold increase in Cu content and a twofold decrease in Fe content compared with cells maintained in 0.4 microM Cu. (64)Cu uptake in Cu-deficient Hep-G2 cells showed a twofold decrease in K(m) compared with cells grown in 10 microM Cu. The decreased K(m) may represent an adaptive response to Cu deficiency. Cells treated with >50 microM Cu, showed an eightfold increase in cytosolic metallothionein. DMT1 protein decreased (35%), suggesting that intracellular Cu caused a reduction of DMT1 protein levels. Our data indicate that, as a result of Cu overload, Hep-G2 cells reduced their Fe content and their DMT1 protein levels. These findings strongly suggest a relationship between Cu and Fe homeostasis in Hep-G2 cells in which Cu accumulation downregulates DMT1 activity.     

Acyclonucleoside iron chelators of 1-(2-hydroxyethoxy)methyl-2-alkyl-3-hydroxy-4-pyridinones: potential oral iron chelation therapeutics

The method of synthesizing acyclonucleoside iron chelators is both convenient and cost effective compared to that of synthesizing ribonucleoside iron chelators. The X-ray crystal structural analysis shows that the 2-hydroxyethoxymethyl group does not affect the geometry of the iron chelating sites. Therefore, the iron binding and removal properties of the acyclonucleoside iron chelators should remain similar to the ribonucleoside iron chelators, which is confirmed by the titration and competition reaction of the acyclonucleoside chelators with iron and ferritin, respectively. The acyclonucleoside iron chelators are more lipophilic with measured n-octanol and Tris buffer distribution coefficients than ribonucleoside iron chelators.