Ubiquitination capabilities in response to neocarzinostatin and H2O2 stress in cell lines from patients with ataxia-telangiectasia

Abstract

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 in¯uenced 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 H 2 O 2 as the stress, in view of preexisting evidence of the e ects of H 2 O 2 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 H 2 O 2 . Both cell types showed a rapid 50 ± 150% increase in de novo formed 125 I-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 aǹ aged' phenotype. The indication that ubiquitin conjugate levels and ubiquitin conjugation capabilities are enhanced upon oxidative stress without signi®cant 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.

Glycation‐altered proteolysis as a pathobiologic mechanism that links dietary glycemic index, aging, and age‐related disease (in nondiabetics)

“…Our finding increased levels of ubiquitin conjugates in many age-related debilities, when cells confront chronic stress (Taylor et al , 2002), and in glycatively stressed RPE may appear counter intuitive if glycation or oxidation diminishes the efficacy of individual components of the ubiquitination machinery (Lowe & Mayer, 1989; Jahngen-Hodge et al , 1992). It is noteworthy that the more AGEs and ubiquitin conjugates accumulate, the greater the proportion of these moieties that is directed to the insoluble fraction.…”

Oxidative stress, mitochondrial abnormalities and antioxidant defense in Ataxia-telangiectasia, Bloom syndrome and Nijmegen breakage syndrome

“…IR, hydrogen peroxide (H 2 O 2 ), nitric oxide (NO) as well as t -butyl hydroperoxide [7], [40], [41]) resulting in oxidative stress and oxidative damage to important biomacromolecules and cell structures [42]. In contrast to exogenous factors, the well-established endogenous ROS-generating sources include the metabolism of cytochrome P450, microsomes and peroxisomes as well as the activities of xanthine oxidase (XO), NADPH oxidase, lipoxygenases (LOX) and peroxidases (Px) [28], [31].…”

“…They belong to a group of chromosomal breakage syndromes (CBS) and are characterised by an increased rate of chromosomal rearrangements and genomic instability, which may arise either spontaneously or as a consequence of exposition to various DNA-damaging factors such as ionising radiation (IR), external radiomimetic chemicals and/or ultraviolet (UV) light [3], [4], [5], [6], [7], [8]. Indeed, cells obtained from A-T, BS and NBS patients show signs of DNA injury, cytoskeletal and chromatin reorganisation, defective or excessive apoptotic cell death and aberrant cell cycle checkpoint function [3], [5], [9], [10], [11].…”

Ubiquitin–proteasome pathway and cellular responses to oxidative stress

“…Thus, accumulation of ubiquitin conjugates in cells or tissues may indicate mild oxidative stress. Importantly, the increased levels of ubiquitin conjugates can be detected prior to changes in GSSG/GSH ratio or other oxidative markers, such as protein carbonyls [198], making the accumulation of ubiquitin conjugates among the most sensitive indicators of cellular oxidative stress. In contrast, extensive oxidative stress may reduce the levels of ubiquitin conjugates in cells due to inactivation of the ubiquitin conjugating enzymes [150, 151].…”