Derivatization of gamma-glutamyl semialdehyde residues in oxidized proteins by fluoresceinamine

Oxidative modification of proteins is implicated in a number of physiologic and pathologic processes. Metal-catalyzed oxidative modification usually causes inactivation of enzymes and the appearance of carbonyl groups in amino acid side chains of the protein. We describe use of fluoresceinamine to label certain of those carbonyl groups. Fluoresceinamine reacted with those carbonyl groups to form a Schiff base which was reduced by cyanoborohydride to yield a stable chromophore on the oxidized residue. The high molar absorbtivity of the fluorescein moiety conferred high sensitivity upon the method. Labeled peptides were readily identified after tryptic digestion of oxidized glutamine synthetase. Further, acid hydrolysis of labeled glutamine synthetase allowed isolation of the derivatized, oxidized residue. The oxidized amino acid was identified as gamma-glutamyl semialdehyde. During metal-catalyzed oxidation, the inactivation of glutamine synthetase paralleled the appearance of gamma-glutamyl semialdehyde.     

Serial protein labeling with infrared maleimide dyes to identify cysteine modifications

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification and quantification of growing importance. However, saturation labeling of thiols with fluorescent dyes results in poor protein recuperation and therefore requires the use of large quantities of starting material. This is especially important in sequential dye-labeling steps when applied for an identification of cysteine modifications. First, we studied the effects of different detergents during labeling procedure, i.e. Tween 20, Triton X-100 and CHAPS, on protein yield and composition. Tween 20 and Triton X-100 resulted in yields of around 50% labeled proteins compared to only 10% with PBS alone and a most diversified 2-DE protein pattern. Secondly, Tween 20 was used for serial protein labeling with maleimid fluorophores, first to conjugate to accessible thiols and after a reduction to label with another fluorophore previously masked di-sulphide and/or oxidized proteins in frontal cortex autopsy tissue of a subject with mild Alzheimer's disease. Two-DE DIGE revealed a complex protein pattern of readily labeled thiols and di-sulphide and/or oxidized proteins. Seventeen proteins were identified by MALDI-TOF and by peptide fingerprints. Several proteins were oxidized and involved in Alzheimer's disease. However methionine oxidation was prevalent. Infrared DIGE may provide an additional tool for an identification of oxidation susceptible proteins.     

Free radical-mediated oxidation of free amino acids and amino acid residues in proteins

Summary. We summarize here results of studies designed to elucidate basic mechanisms of reactive oxygen (ROS)-mediated oxidation of proteins and free amino acids. These studies have shown that oxidation of proteins can lead to hydroxylation of aromatic groups and aliphatic amino acid side chains, nitration of aromatic amino acid residues, nitrosylation of sulfhydryl groups, sulfoxidation of methionine residues, chlorination of aromatic groups and primary amino groups, and to conversion of some amino acid residues to carbonyl derivatives. Oxidation can lead also to cleavage of the polypeptide chain and to formation of cross-linked protein aggregates. Furthermore, functional groups of proteins can react with oxidation products of polyunsaturated fatty acids and with carbohydrate derivatives (glycation/glycoxidation) to produce inactive derivatives. Highly specific methods have been developed for the detection and assay of the various kinds of protein modifications. Because the generation of carbonyl derivatives occurs by many different mechanisms, the level of carbonyl groups in proteins is widely used as a marker of oxidative protein damage. The level of oxidized proteins increases with aging and in a number of age-related diseases. However, the accumulation of oxidized protein is a complex function of the rates of ROS formation, antioxidant levels, and the ability to proteolytically eliminate oxidized forms of proteins. Thus, the accumulation of oxidized proteins is also dependent upon genetic factors and individual life styles. It is noteworthy that surface-exposed methionine and cysteine residues of proteins are particularly sensitive to oxidation by almost all forms of ROS; however, unlike other kinds of oxidation the oxidation of these sulfur-containing amino acid residues is reversible. It is thus evident that the cyclic oxidation and reduction of the sulfur-containing amino acids may serve as an important antioxidant mechanism, and also that these reversible oxidations may provide an important mechanism for the regulation of some enzyme functions.     

Age-related changes in the expression and oxidation of bronchoalveolar lavage proteins in the rat

The incidence and severity of many lung diseases change with age. Some diseases, such as pneumonia, occur with increased frequency in children and the elderly. Proteins obtained by bronchoalveolar lavage (BAL) serve as the first line of defense against inhaled toxins and pathogens. Age-related changes in BAL protein expression and oxidative modification were examined in juvenile (1 mo), young adult (2 mo), and aged (18 mo) F344 rats using two-dimensional difference gel electrophoresis (2D-DIGE), matrix-assisted laser desorption ionization-time of flight/time of flight (MALDI-ToF/ToF) tandem mass spectrometry, and carbonyl immunoblotting. Using 2D-DIGE, we detected 563 protein spots, and MALDI-ToF/ToF identified 204 spots comprising 31 proteins; 21 changed significantly (17 increases) between juvenile and young adult or aged rats, but for 12 of these proteins, levels had a biphasic pattern, and levels in aged rats were less than in young adults. Relative carbonylation was determined by comparison of immunostaining with total protein staining on each oxidized protein blot. We found that aged rats had significantly increased oxidation in 13 proteins compared with juvenile rats. Many of the proteins altered in expression or oxidation level had functions in host defense, redox regulation, and protein metabolism. We speculate that low levels of expression of host defense proteins in juvenile rats and decreases in levels of these proteins between young adult and aged rats may predispose these groups to pneumonia. In addition, we have shown age-related increases in protein oxidation that may compromise host defense function in aged rats.     

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.     

Proteomic analysis of redox- and ErbB2-dependent changes in mammary luminal epithelial cells using cysteine- and lysine-labelling two-dimensional difference gel electrophoresis

Differential protein expression analysis based on modification of selected amino acids with labelling reagents has become the major method of choice for quantitative proteomics. One such methodology, two-dimensional difference gel electrophoresis (2-D DIGE), uses a matched set of fluorescent N-hydroxysuccinimidyl (NHS) ester cyanine dyes to label lysine residues in different samples which can be run simultaneously on the same gels. Here we report the use of iodoacetylated cyanine (ICy) dyes (for labelling of cysteine thiols, for 2-D DIGE-based redox proteomics. Characterisation of ICy dye labelling in relation to its stoichiometry, sensitivity and specificity is described, as well as comparison of ICy dye with NHS-Cy dye labelling and several protein staining methods. We have optimised conditions for labelling of nonreduced, denatured samples and report increased sensitivity for a subset of thiol-containing proteins, allowing accurate monitoring of redox-dependent thiol modifications and expression changes. Cysteine labelling was then combined with lysine labelling in a multiplex 2-D DIGE proteomic study of redox-dependent and ErbB2-dependent changes in epithelial cells exposed to oxidative stress. This study identifies differentially modified proteins involved in cellular redox regulation, protein folding, proliferative suppression, glycolysis and cytoskeletal organisation, revealing the complexity of the response to oxidative stress and the impact that overexpression of ErbB2 has on this response.     

Apolipoprotein E genotype affects tissue metallothionein levels: studies in targeted gene replacement mice

The apolipoprotein E (APOE) genotype is an important risk factor for ageing and age-related diseases. The APOE4 genotype (in contrast to APOE3) has been shown to be associated with oxidative stress and chronic inflammation. Metallothioneins (MT) exhibit antioxidant and anti-inflammatory activity, and MT overexpression has been shown to increase lifespan in mice. Interactions between APOE and MT, however, are largely unknown. Hence, we determined the effect of the APOE4 versus APOE3 genotype on MT levels in targeted gene replacement mice. APOE4 versus APOE3 mice exhibited significantly lower hepatic MT1 and MT2 mRNA as well as lower MT protein levels. The decrease in hepatic MT protein levels in APOE4 as compared to APOE3 mice was accompanied by lower nuclear Nrf1, a protein partly controlling MT gene expression. Cell culture experiments using hepatocytes identified allyl-isothiocyanate (AITC) as a potent MT inductor in vitro. Therefore, we supplemented APOE3 and APOE4 mice with AITC. However, AITC (15 mg/kg b.w.) could only partly correct for decreased MT1 and MT2 gene expression in APOE4 mice in vivo. Furthermore, cholesterol significantly decreased both Nrf1 and MT mRNA levels in Huh7 cells indicating that differences in MT gene expression between the two genotypes could be related to differences in hepatic cholesterol concentrations. Overall, present data suggest that the APOE genotype is an important determinant of tissue MT levels in mice and that MT gene expression may be impaired by the APOE4 genotype.     

Progression and specificity of protein oxidation in the life cycle of Arabidopsis thaliana

Protein carbonylation is an irreversible oxidative process leading to a loss of function of the modified proteins, and in a variety of model systems, including worms, flies, and mammals, carbonyl levels gradually increase with age. In contrast, we report here that in Arabidopsis thaliana an initial increase in protein oxidation during the first 20 days of the life cycle of the plant is followed by a drastic reduction in protein carbonyls prior to bolting and flower development. Protein carbonylation prior to the transition to flowering targets specific proteins such as Hsp70, ATP synthases, the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), and proteins involved in light harvesting/energy transfer and the C2 oxidative photosynthetic carbon cycle. The precipitous fall in protein carbonyl levels is due to the specific reduction in the levels of oxidized proteins rather than an overall loss of chlorophyll and Rubisco associated with the senescence syndrome. The results are discussed in light of contemporary theories of aging in animals.     

Apolipoprotein e4 impairs macrophage efferocytosis and potentiates apoptosis by accelerating endoplasmic reticulum stress

Apolipoprotein (apo) E4 is a major genetic risk factor for a wide spectrum of inflammatory metabolic diseases, including atherosclerosis, diabetes, and Alzheimer disease. This study compared diet-induced adipose tissue inflammation as well as functional properties of macrophages isolated from human APOE3 and APOE4 mice to identify the mechanism responsible for the association between apoE4 and inflammatory metabolic diseases. The initial study confirmed previous reports that APOE4 gene replacement mice were less sensitive than APOE3 mice to diet-induced body weight gain but exhibited hyperinsulinemia, and their adipose tissues were similarly inflamed as those in APOE3 mice. Peritoneal macrophages isolated from APOE4 mice were defective in efferocytosis compared with APOE3 macrophages. Increased cell death was also observed in APOE4 macrophages when stimulated with LPS or oxidized LDL. Western blot analysis of cell lysates revealed that APOE4 macrophages displayed elevated JNK phosphorylation indicative of cell stress even under basal culturing conditions. Significantly higher cell stress due mainly to potentiation of endoplasmic reticulum (ER) stress signaling was also observed in APOE4 macrophages after LPS and oxidized LDL activation. The defect in efferocytosis and elevated apoptosis sensitivity of APOE4 macrophages was ameliorated by treatment with the ER chaperone tauroursodeoxycholic acid. Taken together, these results showed that apoE4 expression causes macrophage dysfunction and promotes apoptosis via ER stress induction. The reduction of ER stress in macrophages may be a viable option to reduce inflammation and inflammation-related metabolic disorders associated with the apoE4 polymorphism.     

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Apolipoprotein-D expression is increased during development and maturation of the human prefrontal cortex

Apolipoprotein D (apoD) is a lipid binding protein expressed in the brain where its function is largely unknown. Based on changes in lipid metabolism and deposition that occur in the human brain during postnatal development, we investigated changes in apoD expression in the prefrontal cortex in 69 normal cases ranging in age from 40 days to 49 years utilizing gene microarray, quantitative PCR and western blotting methods. In contrast to the high expression of apolipoprotein E ( APOE ), low-density lipoprotein receptor-related protein 8 ( LRP8 ) and 3-hydroxy-3-methyl-glutaryl-CoA reducatase ( HMGCR ) (genes that play a role in lipid-related pathways in brain development) early in life, apoD expression was low in neonates and increased in expression throughout life resulting in six- to eight-fold higher levels at the mRNA and protein levels in adults. Recent studies suggest that apoD has a novel antioxidant function in the brain and we found that the increased apoD expression throughout development and into adulthood was correlated with the expression of antioxidant genes superoxide dismutase 1 ( SOD1 ) and glutathione peroxidase 3 ( GPX3 ) as well as proteins that were modified by the lipid peroxidation end-product 4-hydroxynonenal. These studies reveal that apoD expression is increased throughout life in the human prefrontal cortex and that this is correlated with genetic and biochemical markers of oxidative stress.     

Quantitative proteome analysis of age‐related changes in mouse gastrocnemius muscle using mTRAQ

Aging is associated with a progressive loss of skeletal muscular function that often leads to progressive disability and loss of independence. Although muscle aging is well documented, the molecular mechanisms of this condition still remain unclear. To gain greater insight into the changes associated with aging of skeletal muscle, we performed quantitative proteomic analyses on young (6 months) and aged (27 months) mouse gastrocnemius muscles using mTRAQ stable isotope mass tags. We identified and quantified a total of 4585 peptides corresponding to 236 proteins (protein probability >0.9). Among them, 33 proteins were more than 1.5‐fold upregulated and 20 proteins were more than 1.5‐fold downregulated in aged muscle compared with young muscle. An ontological analysis revealed that differentially expressed proteins belonged to distinct functional groups, including ion homeostasis, energy metabolism, protein turnover, and Ca²⁺ signaling. Identified proteins included aralar1, β‐enolase, fatty acid‐binding protein 3, 3‐hydroxyacyl‐CoA dehydrogenase (Hadh), F‐box protein 22, F‐box, and leucine‐rich repeat protein 18, voltage‐dependent L‐type calcium channel subunit beta‐1, ryanodine receptor (RyR), and calsequestrin. Ectopic expression of calsequestrin in C2C12 myoblast resulted in decreased activity of nuclear factor of activated T‐cells and increased levels of atrogin‐1 and MuRF1 E3 ligase, suggesting that these differentially expressed proteins are involved in muscle aging.     

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.     

APOE genotype affects the pre‐synaptic compartment of glutamatergic nerve terminals

Apolipoprotein E (APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock‐in mice, we have previously shown that APOE‐ε4 Targeted Replacement (TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post‐synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate–glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE‐ε3 TR mice, APOE‐ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE‐ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre‐synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4–5 months and 1 year. Using high‐frequency ¹³C/¹H nuclear magnetic resonance spectroscopy, we found that APOE‐ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE‐ε4, and also act as surrogate markers for Alzheimer's disease risk.     

Identification of oxidized plasma proteins in Alzheimer's disease

The modification of proteins by reactive oxygen species is central to the pathology of Alzheimer's disease (AD). Previously, we have observed specific oxidized proteins in blood plasma of AD subjects [Biochem. Biophys. Res. Commun. 275 (2000) 678]. Plasma from AD subjects and age-matched controls was subjected to two-dimensional gel electrophoresis (2-DE). Oxidized proteins with new carbonyl groups were detected by reaction with 2,4-dinitrophenylhydrazine, followed by Western blotting with anti-DNP antibody. Seven principal oxidized protein spots (isoelectric point=4.7-5.5; molecular mass=45-65 kDa) were observed, with varying levels of oxidation in plasma samples from both AD and non-AD subjects. Matrix-assisted laser desorption mass spectroscopy (MALDI-TOF/MS) revealed that these oxidized proteins were isoforms of fibrinogen gamma-chain precursor protein and of alpha-1-antitrypsin precursor. These proteins exhibited a two- to sixfold greater specific oxidation index in plasma from AD subjects when compared to controls. Both these proteins have been previously implicated in the pathology of the disease. It is possible that oxidized isoforms of these proteins may serve as biomarkers for AD.     

Differential Proteomics Analysis of Specific Carbonylated Proteins in the Temporal Cortex of Aged Rats: The Deterioration of Antioxidant System

Oxidative stress plays a pivotal role in normal brain aging and various neurodegenerative diseases, including Alzheimer’s disease (AD). Irreversible protein carbonylation, a widely used marker for oxidative stress, rises during aging. The temporal cortex is essential for learning and memory and particularly susceptible to oxidative stress during aging and in AD patients. In this study, we used 2-DE, MALDI-TOF/TOF MS, and Western blotting to analyze the differentially carbonylated proteins in the rat temporal cortex between 1-month-old and 24-month-old. We showed that the carbonyl levels of ten protein spots corresponding to six gene products: SOD1, SOD2, peroxiredoxin 1, peptidylprolyl isomerase A, cofilin 1, and adenylate kinase 1, significantly increased in the temporal cortex of aged rats. These proteins are associated with antioxidant defense, the cytoskeleton, and energy metabolism. Several oxidized proteins identified in aged rat brain are known to be involved in neurodegenerative disorders as well. Our findings indicate that these carbonylated proteins may be implicated in the decline of normal brain aging process and provide insights into the mechanisms underlying age-associated dysfunction of temporal cortex.     

Protein oxidation and proteolysis during aging and oxidative stress

Previous studies in this laboratory have shown that glutamine synthetase (GS) and other key metabolic enzymes are inactivated by metal-catalyzed oxidation reactions in vitro. Oxidative inactivation renders these proteins highly susceptible to proteolysis, especially to a class of newly identified alkaline proteases which exhibit little or no activity against the native enzymes. These studies have suggested that oxidative inactivation may be an important marking step for intracellular protein degradation. Because many of the enzymes which have been shown to accumulate as inactive or less active forms during aging are readily inactivated by metal-catalyzed oxidation reactions in vitro, we have investigated the possible relationship between protein oxidation and proteolysis during aging and oxidative stress in vivo. Oxidized proteins accumulate in hepatocytes of rats exposed to 100% oxygen during the first 48 h of oxygen treatment. In the interval between 48 and 54 h the levels of oxidized proteins decline sharply. The specific activities of at least two liver enzymes, glutamine synthetase and glucose-6-phosphate dehydrogenase (G-6-PDH), decrease during the 54-h experiment. GS and G-6-PDH specific immunological cross-reactivity remains high during the first 48 h of oxygen treatment and then declines in the interval between 48 and 54 h. During this same interval the levels of alkaline proteases which degrade oxidized proteins increase, indicating that these activities are induced or activated in response to oxidative stress and subsequently degrade the proteins which have become oxidized during the initial phase of oxygen treatment. Oxidized proteins accumulate progressively during aging in hepatocytes from rats 3 to 26 months old, with the largest incremental increase between 20 and 26 months. The increase in protein oxidation is correlated with a loss of specific activity of GS and G-6-PDH without a concomitant loss of immunological cross-reactivity. The levels of alkaline proteases which degrade oxidized proteins in hepatocytes from 26-month-old rats is only 20% that of 3-month-old rats, suggesting that oxidized proteins accumulate in hepatocytes from old rats, in part, because the proteases which degrade them are deficient or defective. moreover, when old rats are subjected to treatment with 100% oxygen, the levels of oxidized proteins continue to increase and the alkaline protease activity remains low, indicating that these protease activities are not increased in response to oxidative stress in old rats.     

Increased protein hydrophobicity in response to aging and Alzheimer disease

Increased levels of misfolded and damaged proteins occur in response to brain aging and Alzheimer disease (AD), which presumably increase the amount of aggregation-prone proteins via elevations in hydrophobicity. The proteasome is an intracellular protease that degrades oxidized and ubiquitinated proteins, and its function is known to be impaired in response to both aging and AD. In this study we sought to determine the potential for increased levels of protein hydrophobicity occurring in response to aging and AD, to identify the contribution of proteasome inhibition to increased protein hydrophobicity, and last to identify the contribution of ubiquitinated and oxidized proteins to the pool of hydrophobic proteins. In our studies we identified that aging and AD brain exhibited increases in protein hydrophobicity as detected using Bis ANS, with dietary restriction (DR) significantly decreasing age-related increases in protein hydrophobicity. Affinity chromatography purification of hydrophobic proteins from aging and AD brains identified increased levels of oxidized and ubiquitinated proteins in the pool of hydrophobic proteins. Pharmacological inhibition of the proteasome in neurons, but not astrocytes, resulted in an increase in protein hydrophobicity. Taken together, these data indicate that there is a relationship between increased protein oxidation and protein ubiquitination and elevations in protein hydrophobicity within the aging and the AD brain, which may be mediated in part by impaired proteasome activity in neurons. Our studies also suggest a potential role for decreased oxidized and hydrophobic proteins in mediating the beneficial effects of DR.     

Apolipoprotein E genotyping among the healthy Kuwaiti population

Apolipoproteins (lipid-free) are lipid-binding proteins that circulate in the plasma of human blood and are responsible for the clearance of lipoproteins. Apolipoprotein E (ApoE) is one of the several classes of this protein family. It acts as a ligand for the low-density lipid (LDL) receptors and is important for the clearance of very low-density lipid (VLDL) and chylomicron remnants. The APOE gene locus is polymorphic, with three major known alleles, APOE*3, *4, and *2. We investigated the distribution of the allele frequency of the APOE gene locus and describe here the genetic variation in four Kuwaiti subpopulations: Arab origin (Arabian peninsula), Arab Bedouin tribes, Iranian origin, and the heterogeneous population. We also describe the use of Spreadex gels in resolving the amplified and digested products of the APOE gene locus. DNA was extracted from whole blood and subjected to PCR and then to RFLP analysis. Allele and genotype frequencies were estimated for the total population and for each subpopulation. Statistical analysis showed no difference in the allele frequencies between the four groups. The frequency of APOE*3 in the Kuwaiti population was highest (88.4%) followed by the frequency of APOE*4 (6.5%) and APOE*2 (5.1%). The genotype and allele frequencies obtained for the Kuwaiti population fell within the reported worldwide distribution for the APOE gene locus. Moreover, the results obtained in this study showed no statistical difference (p > 0.05) between the APOE allele and genotype frequencies between the subgroups for all six genotypes and three alleles, supporting the assumption of admixture in the Kuwaiti population and that the obtained frequencies were in Hardy-Weinberg equilibrium. Finally, we found that the distribution of the APOE alleles in Kuwait differs somewhat from those reported in other Arab populations, suggesting that the Arabs originating from the Arabian peninsula are different from those of Lebanon, Morocco, and Sudan.