alpha-chymotrypsin and 4-hydroxy-2-nonenal

The proteasome plays a fundamental role in processes essential for cell viability. A loss in proteasome function has been associated with aging, as well as a number of age-related diseases. Defining the mechanism(s) behind this loss in function will add important information regarding the molecular basis for aging. In the current study, we performed an age-based comparison of proteasome function and composition of subunits and regulatory proteins in the neural retina and retinal pigment epithelium (RPE) in Fischer 344 rats. In the RPE, there was no age-dependent difference in activity, subunit composition, or content of proteasome regulators, PA28 and PA700. In contrast, the aged neural retina demonstrated a significant reduction in the chymotrypsin-like activity and decreased degradation of both casein and casein modified by 4-hydroxynonenal. This loss in function could not be explained by differences in subunit composition, content of PA28 and PA700, or reversible modification of cysteine residues. To begin investigating the molecular basis for the age-associated decrement in proteasome function, we modified the cysteine residues in proteasome from young rats with the sulfhydryl-reactive chemical N-ethylmaleimide. We observed inhibition of the chymotrypsin-like activity and decreased degradation of casein that was comparable to that seen in aged retinas. Thus, chemical modification of cysteine provides an in vitro method that partially recapitulates aging proteasome. Further studies are required to confirm irreversible modification of functionally significant cysteine as a potential mechanism behind the age-related loss in proteasome function.

Topics: Aging; Aldehydes; Animals; Caseins; Chelating Agents; Chymotrypsin; Cross-Linking Reagents; Enzyme Inhibitors; Ethylmaleimide; Eye Proteins; HSP90 Heat-Shock Proteins; Pigment Epithelium of Eye; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Denaturation; Rats; Rats, Inbred F344; Retina

The role of oxidative stress in alcoholic liver disease and cytokeratin aggresome formation is the focus of this in vitro study. HepG2 cells transduced to over express CYP2E1 (E47) and control HepG2 cells (C34) were first treated with arachidonic acid, then Fe-NAT, and finally with ethanol. In the E47 ethanol-treated cells, CYP2E1 was induced and a higher level of reactive oxygen species and carbonyl proteins were generated. The proteasome activity decreased significantly in the E47 ethanol-treated cells. This inhibition was prevented when CYP2E1 was inhibited by DAS. Microarray analysis showed gene expression down regulation of the proteasome subunit, as well as ubiquitin pathway proteins in the E47 ethanol-treated cells. 4-Hydroxynonenal (4-HNE) adducts were increased in the E47 cells treated with ethanol. Furthermore, the immunoprecipitated 4-HNE modified proteins from these cells stained positive with antibodies to the proteasome subunit alpha 6. These results indicate that the ethanol induced CYP2E1 generates oxidative stress that is responsible for the decrease in proteasome activity. Cytokeratin 8 and 18 were induced by ethanol treatment of E47 cells and polyubiquitinated forms of these proteins were found in the polyubiquitin smear upon Western blots analysis. Cytokeratin aggresomes and Mallory body-like inclusions formed in the ethanol-treated E47 cells, indicating that the ubiquitinated cytokeratins accumulated as a result of the inhibition of the proteasome by ethanol treatment when oxidation of ethanol induced oxidative stress. This is the first report where ethanol caused Mallory body-like cytokeratin inclusions in transformed human liver cells in vitro.

Topics: Aldehydes; Allyl Compounds; Arachidonic Acid; Cell Survival; Chymotrypsin; Cytochrome P-450 CYP2E1; DNA Damage; Down-Regulation; Enzyme Induction; Ethanol; Fluorescent Antibody Technique; Humans; Inclusion Bodies; Iron; Keratin-18; Keratin-8; Oxidative Stress; Polyubiquitin; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Carbonylation; Reactive Oxygen Species; Sulfides; Up-Regulation

Insulysin (IDE) and neprilysin (NEP) were found to be inactivated by oxidation with hydrogen peroxide, an iron-ascorbate oxidation system, and by treatment with 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). In each case reaction led to the introduction of protein carbonyl groups as judged by reaction with 2,4-dintrophenylhydrazine. IDE was inactivated by reaction with 4-hydroxy-2-nonenal (HNE) with the concomitant formation of protein adducts. NEP was not inactivated to a significant extent by HNE, but some HNE-adduct formation did occur. Prior reaction with hydrogen peroxide or AAPH led to enhanced formation of HNE adducts. Treatment of IDE with AAHP or hydrogen peroxide increased its susceptibility to proteolysis, while treatment of NEP with iron/ascorbate or hydrogen peroxide increased its susceptibility to proteolysis. Since IDE and NEP play a prominent role in the clearance of amyloid beta peptides, their oxidative inactivation and enhanced proteolysis can contribute to the onset and/or progression of Alzheimer's disease.

Topics: Aldehydes; Alzheimer Disease; Amidines; Amyloid beta-Peptides; Ascorbic Acid; Chlorides; Chymotrypsin; Ferric Compounds; Hydrogen Peroxide; Insulysin; Neprilysin; Oxidation-Reduction; Trypsin

The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE.

Topics: Aldehydes; Amino Acid Sequence; Animals; Antioxidants; Binding Sites; Blotting, Western; Catalysis; Chymotrypsin; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Electrophoresis, Gel, Two-Dimensional; Kinetics; Liver; Mass Spectrometry; Molecular Sequence Data; Peptide Mapping; Proteasome Endopeptidase Complex; Rats; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Trypsin

The modification of proteins by 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) was investigated using mass spectroscopic approaches. Electrospray ionization MS analysis of HNE- and ONE-treated myoglobin and apomyoglobin revealed that the latter more "open" protein structure resulted in more extensive modification. Reductive methylation of Lys residues halved the extent of modification, implicating the importance of adduction of HNE and ONE to both His and Lys residues. HPLC-MS/MS analysis of tryptic and chymotryptic peptides of HNE- or ONE-adducted apomyoglobin was aided by the knowledge of structures previously elucidated through model reactions. In the case of HNE, the adducts detected were the HNE-His Michael adduct (on H24, H36, H64, and H113), its dehydrated form (on H36), and the HNE-Lys pyrrole adduct (on K16, K42, K45, K145, and K147). In the case of the more reactive ONE, the adducts detected were the ONE-His Michael adduct (on H24), the ONE-Lys pyrrolinone adduct (on K16 and K145), and the ONE-His-Lys pyrrole cross-link (linking K16 to H24 in the C(5) peptide). Although previous analyses of tryptic peptides yielded findings about the nature of His modification, the current chymotryptic peptide analysis produced the first structural characterization of Lys modification on intact proteins by HNE and ONE using mass spectrometry.

Topics: Aldehydes; Amino Acid Sequence; Apoproteins; Chymotrypsin; Cross-Linking Reagents; Mass Spectrometry; Molecular Sequence Data; Myoglobin; Peptide Fragments; Proteins; Pyrroles; Trypsin

The proteasomal pathway is responsible for processes essential for cell viability, including the selective degradation of oxidized proteins. An age-dependent loss in proteasome function has been reported in many tissues, but has not been examined in the retina. In this study, we evaluated proteasome function and protein oxidation in retinal homogenates from young adult and old F344BN rats. For retinal proteasome from old rats, we observed an 80% decrease in the rate of casein degradation and a 75% loss in chymotrypsin-like activity. This loss in activity could be partially accounted for by a 50% reduction in expression of the 20S proteasome. The regulatory complex PA700 and the inducible beta-subunit, LMP7, which is associated with the chymotrypsin-like activity, were expressed in equivalent concentrations relative to the 20S catalytic core in both young and old rats. Immunochemical analysis using antibodies that recognize the protein oxidative modifications, nitrotyrosine and 4-hydroxy-2-nonenal, showed that retinal proteins from old rats exhibited the greatest immunoreactivity. These results suggest that the age-related loss in proteasome function contributes to the accumulation of oxidized retinal proteins. Thus, the combined effect of an increase in oxidized proteins and inactivation of the protease responsible for ridding the cell of oxidized proteins places the aged retina at greater risk for irreversible damage caused by oxidative stress.

Topics: Aging; Aldehydes; Animals; Blotting, Western; Caseins; Chymotrypsin; Cysteine Endopeptidases; Eye Proteins; Male; Multienzyme Complexes; Oxidation-Reduction; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Inbred BN; Rats, Inbred F344; Retina; Tyrosine

Moderate daily exercise is known to be beneficial to health, reducing risks of a number of age-related disorders. Molecular mechanisms that bring about these effects are not clear. In contrast, it has been claimed that some types of prolonged physical exertion are detrimental to health because active oxygen species are generated excessively by enhanced oxygen consumption. Using two age groups of rats, young (4 week) and middle aged (14 months), we investigated the effects of long-term swimming training on the oxidative status of phospholipids, proteins, and DNA. The concentration of thiobarbituric acid reactive substances and 4-hydroxynonenal protein adducts did not differ in the gastrocnemius muscle between exercised and nonexercised animals in the two age groups. The extent of carbonylation in a protein of molecular weight around 29 KDa and the amount of 8-hydroxydeoxyguanosine in nuclear DNA were smaller (p<.05) in the exercised rats than in the sedentary animals. Activities of DT-diaphorase (C1: 29.3+/-1.9; C2: 36.1+/-2.6; E1: 27.2+/-1.3; C2: 33.4+/-2.9 nmol/mg protein) and proteasome, a major proteolytic enzyme for oxidatively modified proteins were significantly higher in the exercised animals of both age groups (p<.05). The adaptive response against oxidative stress induced by moderate endurance exercise constitutes a beneficial effect of exercise.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Aldehydes; Animals; Chymotrypsin; Cysteine Endopeptidases; Deoxyguanosine; DNA Damage; Male; Multienzyme Complexes; Muscle, Skeletal; NAD(P)H Dehydrogenase (Quinone); Oxidation-Reduction; Peptide Hydrolases; Phospholipids; Physical Exertion; Proteasome Endopeptidase Complex; Proteins; Rats; Rats, Wistar; Swimming; Thiobarbituric Acid Reactive Substances; Trypsin