E. Dudek

Vitamin C and vitamin E restore the resistance of GSH-depleted lens cells to H2O2

A decline in reduced glutathione (GSH) levels is associated with aging and many age-related diseases. The objective of this study was to determine whether other antioxidants can compensate for GSH depletion in protection against oxidative insults. Rabbit lens epithelial cells were depleted of > 75% of intracellular GSH by 25-200 microM buthionine sulfoximine (BSO). Depletion of GSH by BSO alone had little direct effect on cell viability, but resulted in an approximately 30-fold increase in susceptibility to H(2)O(2)-induced cell death. Experimentally enhanced levels of nonprotein sulfhydryls other than GSH (i.e., N-acetylcysteine) did not protect GSH-depleted cells from H(2)O(2)-induced cell death. In contrast, pretreatment of cells with vitamin C (25-50 microM) or vitamin E (5-40 microM), restored the resistance of GSH-depleted cells to H(2)O(2). However, concentrations of vitamin C > 400 microM and vitamin E > 80 microM enhanced the toxic effect of H(2)O(2). Although levels of GSH actually decreased by 10-20% in cells supplemented with vitamin C or vitamin E, the protective effects of vitamin C and vitamin E on BSO-treated cells were associated with significant ( approximately 70%) decreases in oxidized glutathione (GSSG) and concomitant restoration of the cellular redox status (as indicated by GSH:GSSG ratio) to levels detected in cells not treated with BSO. These results demonstrate a role for vitamin C and vitamin E in maintaining glutathione in its reduced form. The ability of vitamin C and vitamin E in compensations for GSH depletion to protect against H(2)O(2)-induced cell death suggests that GSH, vitamin C, and vitamin E have common targets in their actions against oxidative damage, and supports the preventive or therapeutic use of vitamin C and E to combat age- and pathology-associated declines in GSH. Moreover, levels of these nutrients must be optimized to achieve the maximal benefit.

H2O2-mediated oxidative stress activates NF-κB in lens epithelial cells

In the mammalian lens, intracellular oxidants produced by photo-oxidative processes and exposure to toxic chemicals constitute stresses that produce cellular oxidative damage, result in changes in gene expression, and are causally related to cataract formation. Currently, it is believed that H(2)O(2) is the major oxidant to which the lens is exposed. In this report, we examine the activation and regulation of the oxidant-sensitive transcription factor, NF-kappa B, by H(2)O(2)-mediated oxidative stress in lens epithelial cells. Lens epithelial cells treated with H(2)O(2) demonstrated at 1 h a strong activation of NF-kappa B which returned to basal levels by 2 h. Under proteasome inhibition using both MG132 and lactacystin, H(2)O(2)-mediated activation of NF-kappa B was prevented, implicating the involvement of proteasome degradation of I kappa B proteins as being necessary for this activation. However, Western blot analysis demonstrated no degradation of I kappa B-alpha, -beta, or -epsilon associated with H(2)O(2)-mediated NF-kappa B activation. In comparison, when cells were treated with the cytokine TNF-alpha, NF-kappa B was strongly activated and degradation of both I kappa B-alpha and -beta was observed. These results clearly demonstrate that H(2)O(2)-mediated oxidative stress activates NF-kappa B in lens epithelial cells, which may subsequently lead to changes in gene expression. The results also reveal that different signaling pathways in the activation of NF-kappa B in lens epithelial cells are utilized by H(2)O(2) and TNF-alpha. These different pathways of NF-kappa B activation may be required to effect specific NF-kappa B-dependent gene expression in response to these different stimuli.

Novel Control of S Phase of the Cell Cycle by Ubiquitin-conjugating Enzyme H7

Timely degradation of regulatory proteins by the ubiquitin proteolytic pathway (UPP) is an established paradigm of cell cycle regulation during the G2/M and G1/S transitions. Less is known about roles for the UPP during S phase. Here we present evidence that dynamic cell cycle-dependent changes in levels of UbcH7 regulate entrance into and progression through S phase. In diverse cell lines, UbcH7 protein levels are dramatically reduced in S phase but are fully restored by G2. Knockdown of UbcH7 increases the proportion of cells in S phase and doubles the time to traverse S phase, whereas UbcH7 overexpression reduces the proportion of cells in S phase. These data suggest a role for UbcH7 targets in the completion of S phase and entry into G2. Notably, UbcH7 knockdown was coincident with elevated levels of the checkpoint kinase Chk1 but not Chk2. These results argue that UbcH7 promotes S phase progression to G2 by modulating the intra-S phase checkpoint mediated by Chk1. Furthermore, UbcH7 levels appear to be regulated by a UPP. Together the data identify novel roles for the UPP, specifically UbcH7 in the regulation of S phase transit time as well as in cell proliferation.

Perturbing the Ubiquitin Pathway Reveals How Mitosis Is Hijacked to Denucleate and Regulate Cell Proliferation and Differentiation In Vivo

Background The eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6. Methodology and Principal Findings We replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues. Conclusions and Significance K6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.

Ubiquitin Proteasome Pathway–Mediated Degradation of Proteins: Effects Due to Site-Specific Substrate Deamidation

PURPOSE The accumulation, aggregation, and precipitation of proteins is etiologic for age-related diseases, particularly cataract, because the precipitates cloud the lens. Deamidation of crystallins is associated with protein precipitation, aging, and cataract. Among the roles of the ubiquitin proteasome pathway (UPP) is protein surveillance and maintenance of protein quality. The purpose of this study was to determine whether deamidation can alter clearance of crystallins by the UPP. METHODS Wild-type (WT) and deamidated crystallins were expressed and (125)I-radiolabeled. Ubiquitination and degradation were monitored separately. RESULTS For betaB2 crystallins, rates of ubiquitination and adenosine triphosphate-dependent degradation, both indicators of active UPP, occurred in the order Q70E/Q162E>Q162E> Q70E=WT betaB2 using reticulocyte lysate as the source of degradation machinery. Human lens epithelial cell lysates and lens fiber cell lysates also catalyzed ubiquitination but only limited degradation. Supplementation with proteasome failed to enhance degradation. Rates of ubiquitination and degradation of WT and deamidated betaB1 crystallins were rapid and showed little relationship to the site of deamidation using N157D and Q204E mutants. gammaD-Crystallins were not degraded by the UPP. Deamidation altered amine reactivity, circular dichroism spectra, surface hydrophobicity, and thermal stability. CONCLUSIONS These data demonstrate for the first time that, like mild oxidative stress, deamidation of some proteins makes them preferred substrates for ubiquitination and, in some cells, for UPP-dependent degradation. Failure to properly execute ubiquitination and degrade the ubiquitin-conjugates may explain their accumulation on aging and in cataractogenesis.