Robert E. Pacifici

PROTEIN, LIPID AND DNA REPAIR SYSTEMS IN OXIDATIVE STRESS : THE FREE-RADICAL THEORY OF AGING REVISITED

Aerobic organisms are constantly exposed to oxygen radicals and related oxidants. The antioxidant compounds and enzymes they have evolved remove most of the potentially damaging radicals/oxidants; however, damage to cellular proteins, lipids, nucleic acids and carbohydrates can be observed even under normal physiological conditions. Re-reduction of cellular components (direct repair) may be important for some biomolecules. In most cases studied to date, however, enzymatic degradation (by proteases, lipases, nucleases) appears to release damaged elements for excretion and conserve undamaged components for reutilization (indirect repair). In addition, the removal of damaged components appears to prevent or diminish the potential cytotoxicity of oxidized macromolecules. Several studies have reported an accumulation of oxidatively damaged cellular components with age (e.g., cataract formation, lipofuscin). Such reports are evidence that oxidant damage is one of several factors which contribute to the aging process, and provide at least partial support for the free-radical theory of aging. Studies of age-related changes in the activities, or levels of antioxidant enzymes and antioxidant compounds, however, have not provided complete understanding of the putative role of free radicals/oxidants in the aging process. In this review, we present the hypothesis that decreased activities or constitutive levels of oxidant repair enzymes may contribute to a progressive accumulation of oxidant damage with aging. Furthermore, the ability to mount an effective response to oxidative stress (induction of oxidant stress genes and proteins) may decline with age, thus predisposing older cells and organisms to oxidant damage.

[51] Protein degradation as an index of oxidative stress

Publisher Summary This chapter discusses the role of protein degradation as an index of oxidative stress. Oxygen radicals and other activated oxygen species are known to participate in numerous physiological and pathological processes. Situations that augment oxidant exposure, or compromise antioxidant capacity, are commonly referred to as oxidative stress. Oxidative stress can result from exogenous sources or from increases in endogenous oxidative metabolism. Regardless of its source, oxidative stress has been found to affect the behavior of several different cell types. Protein degradation is apparent immediately (no lag phase) after red blood cells (RBC), muscle cells, or bacteria are treated with an oxidant. The response appears to be linear over at least a 3-hr treatment time. Protein degradation assays provide powerful advantages over other techniques. For example, it is possible to perform amino acid analysis to obtain a more complete profile of products. The assays are inherently more sensitive due to the homogeneity of products formed (i.e., amino acids) and the amplification of signal that fluorescence and radiolabeling techniques provide.

Superoxide dismutase is preferentially degraded by a proteolytic system from red blood cells following oxidative modification by hydrogen peroxide.

The cupro-zinc enzyme superoxide dismutase (SOD) undergoes an irreversible (oxidative) inactivation when exposed to its product, hydrogen peroxide (H2O2). Recent studies have shown that several oxidatively modified proteins (e.g., hemoglobin, albumin, catalase, etc.) are preferentially degraded by a novel proteolytic pathway in the red blood cell. We report that bovine SOD is oxidatively inactivated by exposure to H2O2, and that the inactivated enzyme is selectively degraded by proteolytic enzymes in cell-free extracts of bovine erythrocytes. For example, 95% inactivation of SOD by 1.5 mM H2O2 was accompanied by a 106 fold increase in the proteolytic susceptibility of the enzyme during (a subsequent) incubation with red cell extract. Both SOD inactivation and proteolytic susceptibility increased with H2O2 concentration and/or time of exposure to H2O2. Pre-incubation of red cell extracts with metal chelators, serine reagents, or sulfhydryl reagents inhibited the (subsequent) preferential degradation of H2O2-modified SOD. Furthermore, a slight inhibition of degradation was observed with the addition of ATP. We suggest that H2O2-inactivated SOD is recognized and preferentially degraded by the same. ATP-independent, metallo- serine- and sulfhydryl- proteinase pathway which degrades other oxidatively denatured red cell proteins. Related work in this laboratory suggests that this novel proteolytic pathway may actually consist of a 700 kDa enzyme complex of proteolytic activities. Mature red cells have no capacity for de novo protein synthesis but do have extremely high concentrations of SOD. Red cell SOD generates (and is, therefore, exposed to) H2O2 on a continuous basis, by dismutation of superoxide (from hemoglobin autooxidation and the interaction of hemoglobin with numerous xenobiotics).(ABSTRACT TRUNCATED AT 250 WORDS)

ALTERED CELL SURFACE EXPRESSION AND SIGNALING OF LEPTIN RECEPTORS CONTAINING THE FATTY MUTATION

Leptin and the leptin receptor are key players in the regulation of body weight. In an attempt to dissect the molecular mechanism of the Zucker fatty rat leptin receptor mutation (Gln269 → Pro) we analyzed the effects of this mutation on leptin receptor signaling and expression in three different expression systems: 1) 32D cells expressing leptin/erythropoietin receptor chimeras, 2) COS-7 cells expressing a leptin receptor short form, and 3) 293 cells expressing soluble receptor forms. To determine if the Gln269 → Pro mutation is critical for the observed phenotype, we made a similar Gln → Pro mutation at a vicinal residue two amino acids upstream of the fatty mutation to see if it would have similar effects. Incorporation of either of the Gln → Pro mutations into wild type receptor forms did not interfere with leptin binding, but it resulted in a signaling-incompetent receptor. In addition, the majority of the mutant receptor protein was localized intracellularly. Our results suggest that the obese phenotype resulting from the Gln269 → Pro mutation in the leptin receptor of the Zucker fatty rat may be due not only to a reduced cell surface expression of this form of the leptin receptor, but also to a post-leptin binding malfunction of the receptor that interferes with subsequent signal transduction.

Regulation of Gene Expression in Adaptation to Oxidative Stress

Transient adaptation to an acute oxidative stress is a genetically regulated property of facultative bacteria, yeast, and mammalian cells. Experimentally, an initial exposure to sub-lethal concentrations of hydrogen peroxide or superoxide is found to provide significant protection against a subsequent exposure to concentrations that would normally be lethal. Such adaptation is short-term (disappearing within one cell division) and relies upon RNA synthesis and protein synthesis, but does not require DNA replication. Some 30–40 proteins are overexpressed during the bacterial response to hydrogen peroxide or superoxide, and some 20–30 proteins exhibit increased expression in eucaryotes: At least some of these shock or stress proteins are thought to impart the induced resistance to oxidative stress. Four oxidative stress regulons have so far been described in bacteria and at least one antioxidant responsive element is operative in the mammalian genome. Oxidative stress signals appear to be transduced via altered interactions of specific DNA binding proteins with nucleotide target sequences or loci within stress-inducible genes. Efforts are underway to further explain the mechanisms of signal transduction involved in activation of transcription and translation during oxidative stress.