Thomas Nowell

OXIDATIVE STRESS AND ANTIOXIDANT FUNCTION IN RELATION TO RISK FOR CATARACT

Publisher Summary The lens is composed primarily of proteins and water. The light and oxygen to which the lens is exposed are associated with extensive damage to the long-lived lens proteins and other constituents. With progressive damage, the altered proteins accumulate, aggregate, and precipitate in opacities, or cataracts. The young lens has substantial reserves of antioxidants and antioxidant enzymes that may prevent damage. Proteases may selectively remove obsolete proteins and provide a second level of defense. Compromises of function of the lens upon aging are related to deplete or diminished primary antioxidant reserves, antioxidant enzyme capabilities, and diminished secondary defenses such as proteases. Environmental stresses such as smoking and excessive light exposure appear to provide an additional oxidative challenge associated with the depletion of antioxidants as well as with enhanced risk for cataract. This chapter reviews available data and intervention trials, regarding associations between antioxidant nutrients and eye lens cataract in humans. The primary function of the eye lens is to collect and focus light on the retina. The chapter discusses public health issues regarding cataract, clinical features of cataract, role of oxidation and smoking to cataract formation, associations between antioxidants—vitamins C, E, carotenoids, antioxidant nutrient combinations—and cataract, and intervention trial designed to assess the effect of vitamin supplements on cataract risk. Nutrient intake can be optimized to delay cataract. Optimization of nutriture can be achieved through better diets and, with the aid of supplements once appropriate levels of specifically beneficial nutrients are defined. Study in terms of revealing the utility of diet indicates a significant five-fold decrease in risk ratio for cataract between persons consuming ≥1.5 servings of fruits and/or vegetables. It is also essential to know for how long or when would intake of the nutrient be useful with respect to delaying cataract.

Aging, calorie restriction and ubiquitin-dependent proteolysis in the livers of Emory mice

Calorie restriction (R), the only known method to delay the aging process and extend mean and maximal lifespan, has been shown to delay the age-related decline in protein degradation. There are several proteolytic pathways. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) is frequently associated with degradation of damaged abnormal and/or regulatory proteins. We examined the effect of aging and R on supernatants of livers taken from young (4.5 months) and old (23 months) Emory mice. Aging was associated with increased levels of endogenous ubiquitin conjugates, enhanced ability to form high molecular weight conjugates and ubiquitin activating (E1) and ubiquitin conjugating (E2) activity in the control (C) liver supernatants. The age-related increase in levels of endogenous ubiquitin conjugates in liver appears to be primarily due to increased E1 and E2 activities. R prevented the age-related increase in E1 and E2 activity, and thus prevented the age-related increase in levels of ubiquitin conjugates. In spite of the age-related increase in ubiquitin conjugates, no age-related changes in ubiquitin-dependent proteolytic pathway were observed in the C animals. R was associated with an enhanced ability (130%) to degrade beta-lactoglobulin by the ubiquitin-dependent proteolytic pathway in livers from 4.5-month-old animals relative to age-matched C livers. However, rates of the ubiquitin-dependent degradation of beta-lactoglobulin in the 23-month-old C and R animals were indistinguishable. There were no age- or diet-related differences in the ability to degrade another substrate, oxidized ribonuclease (RNase).

Calorie restriction, stress and the ubiquitin-dependent pathway in mouse livers.

Calorie restriction (R) is the only known method to delay the aging process and extend mean and maximal lifespan in rodents. R has been shown to delay the age-related accumulation of damaged proteins and to protect organisms from various stresses which can produce damaged proteins. Such stresses include irradiation, heat shock, and oxidative stress. The ubiquitin- and ATP-dependent proteolytic pathway (UPP) has been associated with the degradation of abnormal and/or damaged proteins. We examined the effect of diet and oxidative stress on activities of the UPP in supernatants from livers taken from 23-month-old Emory mice which had been exposed to an in-vivo injection of paraquat. Paraquat induces oxidative stress by generating superoxide radicals. In livers from non-stressed animals, steady-state levels of endogenous ubiquitin conjugates, de novo conjugate formation, and E1 and E2 activities were significantly lower in R animals than in control (C) animals. However, after exposure to paraquat, levels of endogenous ubiquitin conjugates were significantly higher in R versus C animals, and de novo conjugate formation and E1 and E2 activities in R animals rose to levels which were indistinguishable from levels of these activities noted in C animals. R was associated with an increased ability to degrade beta-lactoglobulin by the UPP after an oxidative stress was imposed. Ability to degrade beta-lactoglobulin by the C or R livers in non-stressed animals was not significantly different. Taken together, these data indicate that oxidative stress in R animals is associated with enhanced levels of ubiquitin conjugates and that this enhancement may be due to an increase in UPP activity. These data also indicate that the ability to form ubiquitin conjugates and the UPP system does not change with oxidative stress in C animals. The latter is consistent with prior reports that suggests that older C animals may already be in a state of enhanced oxidative stress and that activities of the UPP provide a sensitive indicator of levels of cellular redox status.

Ubiquitination capabilities in response to neocarzinostatin and H(2)O(2) stress in cell lines from patients with ataxia-telangiectasia.

The human genetic disorder ataxia-telangiectasia (A-T) is due to lack of functional ATM, a protein kinase which is involved in cellular responses to DNA double strand breaks (DSBs) and possibly other oxidative stresses, as well as in regulation of several fundamental cellular functions. Studies regarding responses in A-T cells to the induction of DSBs utilize ionizing radiation or radiomimetic chemicals, such as neocarzinostatin (NCS), which induce DNA DSBs. This critical DNA lesion activates many defense systems, such as the cell cycle checkpoints. The cell cycle is also regulated through a timed and coordinated degradation of regulatory proteins via the ubiquitin pathway. Our recent studies indicate that the ubiquitin pathway is influenced by the cellular redox status and that it is the major cellular pathway for removal of oxidized proteins. Accordingly, we hypothesized that the absence of a functional ATM protein might involve perturbations to the ubiquitin pathway as well. We show here that upon treatment with NCS, there was a transient 50–70% increase in endogenous ubiquitin conjugates in A-T and wt lymphoblastoid cells. Ubiquitin conjugation capabilities per se and levels of substrates for conjugation were also similarly enhanced in wt and A-T cells upon NCS treatment. We also compared the ubiquitination response in A-T and wt cells using H2O2 as the stress, in view of preexisting evidence of the effects of H2O2 on ubiquitination capabilities in other types of cells. As with NCS treatment, there was an ≈45% increase in endogenous ubiquitin conjugates by 2–4 h after exposure to H2O2. Both cell types showed a rapid 50–150% increase in de novo formed 125I-ubiquitin conjugates. As compared with wt cells, unexposed A-T cells had higher endogenous levels of conjugates and enhanced conjugation capability. However, A-T cells mounted a more muted ubiquitination response to the stress. The enhanced ubiquitin conjugation in unstressed A-T cells and attenuated ability of these cells to respond to stress are consistent with the A-T cells being under oxidative stress and with their having an ‘aged’ phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without significant changes in GSSG/GSH ratios indicates that assays of ubiquitination provide a sensitive measure of cellular stress. The data also add support to the impression that potentiated ubiquitination response to mild oxidative stress is a generalizable phenomenon.

A comparison of ubiquitin-dependent proteolysis of rod outer segment proteins in reticulocyte lysate and a retinal pigment epithelial cell line

We compared ATP- and ubiquitin-dependent proteolysis in supernatants of rabbit reticulocyte lysate and a human retinal pigment epithelial (RPE) cell line. At pH 7.8, both preparations catalyzed the conjugation of [125I]ubiquitin to endogenous proteins, generating an equivalent amount of high mass (> 150 kDa) [125I]ubiquitin-protein adducts. Both preparations degraded exogenous histone 2A, oxRNase and beta-lactoglobulin in an ATP-dependent manner. Addition of ubiquitin (12 or 120 microM) to reticulocyte lysate stimulated (1.4-fold) ATP-dependent degradation only of histone 2A. Addition of 12 microM ubiquitin to RPE supernatant resulted in > or = 3-fold enhancement in degradation of all three substrates. Next, we compared the ability of the two proteolysis systems to degrade bovine rod outer segment (ROS) nonintegral membrane proteins. [125I]ROS protein degradation by reticulocyte lysate was almost exclusively ATP-dependent and was completely inhibited by hemin and vanadate, inhibitors of ATP- and ubiquitin-dependent proteolysis. RPE supernatant also degraded ROS proteins by an ATP-dependent mechanism, and, unlike results obtained in reticulocyte assays, this degradation increased (2-fold) upon ubiquitin supplementation. Both proteolysis systems degraded ROS proteins of molecular mass approximately 10, 30, 37, 40 and 60 kDa, with coincident formation of high mass species. Reticulocyte lysate also degraded 100 and 150 kDa ROS proteins, whereas RPE supernatant did not. The 10, 37 and 40 kDa species were identified by western blot as the gamma-, beta- and alpha- subunits of rod transducin (Gt), respectively. RPE supernatant generated (some) ROS proteolysis products that remained acid-precipitable. As compared with patterns of proteolysis in reticulocytes, RPE supernatant (1) degraded 100% more Gt beta gamma, (2) generated > 10-fold the amount of high mass (putative ubiquitin-ROS protein) conjugates and (3) preferentially degraded Gt beta gamma relative to G t alpha. The ubiquitin-dependent enhancement of ATP-dependent degradation of all proteins tested in RPE supernatant makes the RPE system a valuable experimental tool for the explicit demonstration of ubiquitin-dependent proteolysis.

The effects of aging and calorie restriction on plasma nutrient levels in male and female Emory mice

We examined the effect of diet, age (4.5, 13 and 23 months), and sex on plasma levels of retinol, tocopherol, ascorbate, cholesterol, glucose and glycohemoglobin in male and female Emory mice which were fed control (C) and 50% calorie restricted (R) diets. Results showed that C fed animals tended to have higher levels of plasma ascorbate (50-71%), cholesterol (23-71%), glucose (38-81%) and glycohemoglobin (50%). However, these diet differences varied with the age and sex of the animals. Plasma retinol levels were lower only in R males vs. C males (50%). Novel sex-related differences in levels of plasma retinol (2-fold higher in C male mice than in C or R female mice) are described. Aging was associated with trends towards lower levels of plasma ascorbate (14-25%), glucose (34-36%) and glycohemoglobin (47-57%) from 4.5 to 23 months of age. However, these age differences depended upon the diet and sex of the animals. These data suggest that lower plasma levels of glucose, glycosylated hemoglobin and cholesterol may be causally related to the life extension noted in R animals since elevated levels of these moieties have been related to aging. Since oxidative stress is thought to be causally related to aging it appears unlikely that retinol, tocopherol and ascorbate are causally related to R-induced life-extension.

Structure and Function of Bovine Lens Aminopeptidase and Comparison with Homologous Aminopeptidases

Lens leucine aminopeptidase (LAP†) is the aminopeptidase for which structural, kinetic, and mechanistic information is most developed. Crystallographic, electron micrographic, NMR, and photoaffinity labeling and modeling studies indicate that lens LAP protomers are bilobal, and that bestatin and substrates are bound in an active site, which is found in the larger lobe of each protomer. Zn2+ is involved in substrate liganding and presumably in catalysis of hydrolysis. There is no evidence of an acyl-enzyme intermediate in hydrolysis. Amino acid sequences determined directly or deduced from cDNAs indicate homologies between lens aminopeptidase and APs in organisms as diverse as E. coli, particularly in catalytically important residues, or in residues involved in metal ion binding. For additional details regarding structures and function of some other APs, readers are referred to subsequent chapters in this book.