PDFlib PLOP: PDF Linearization, Optimization, Protection

The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have prevously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation-sensitve enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin-trapping compound N-tert-butyl-phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence-accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8-sensitive line were compared to the SAMR1resistant mice following 14 days of daily PBN treatment at a dose of 30mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These comThis work was supported in part by NIH Grants (AG-10836 and AG-05119). Address correspondence to D. Allan Butterfield, Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, KY 40506-0055. Tel: 606/257-3184; fax: 606/ 257-5876; email: [email protected] BUTTERFIELD et al.: STRUCTURAL AND FUNCTIONAL CHANGES 449 ponents may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging. FREE RADICALS AND OXIDATIVE STRESS The evolutionary process selected oxygen over other gases because of its ready availability, the high energy yield of oxidation, easy distribution in its gaseous state, solubility in biocomponents, and its efficient recycling using the processes of respiration and photosynthesis. Oxygen, however, is also the main source of damaging free radicals, which have been suggested to cause aging and ultimately the death of the organism. There are other sources of free radicals, namely, ionizing radiations like X-rays, ultrasound, photochemical reactions, and biochemical and enzymatic processes; however, the human body is not exposed to all of these as frequently as it is to oxygen-derived radicals. Oxidative injury is the result of an attack on cellular components by highly reactive, toxic oxygen moieties, collectively referred to as reactive oxygen species (ROS). Hydroxyl radicals, peroxyl radicals, superoxide anions, hydrogen peroxide, and nitric oxide are all a part of the ROS family. The half-lives of these free radicals generated in the cell vary from nanoseconds for the highly reactive hydroxyl radical to seconds for nitric oxide and peroxyl radicals. Also the reactivity of these radicals varies from the aqueous environment to those reacting deep within the membrane lipid bilayer. Oxy radicals, like hydroxyl radicals, have a very short life span, are extremely reactive, and hence attack the cellular components present in the vicinity of their production, whereas nitric oxide is very stable and relatively benign, except when it reacts, at diffusion-limited rates, with the superoxide anion to form peroxynitrite. Peroxynitrite is highly reactive and toxic to the cell, affecting several cellular components, leading to loss in structure and function. Hence, the radical damage occurring in the cell is all pervasive. Intracellularly, mitochondria are a major source of free radicals. Normal metabolism in healthy individuals uses the electron transport system in the mitochondria for energy production and in the process gives rise to a host of ROS. There are also various enzymatic and nonenzymatic metal-catalyzed systems capable of generating free radicals. To counteract these damaging free radical species, highly effective antioxidant systems have been developed that include enzymes like glutathione peroxidase, glutathione reductase, S-methyl transferase, superoxide dismutase (SOD), and catalase that can either act as repair agents or as antioxidant enzymes by eliminating precursors like hydrogen peroxide and superoxide from the cellular system. Also, there are proteins like hemoglobin, transferrin, and ceruloplasmin that bind ferrous and copper ions and prevent radical generation through Fenton chemistry; and proteases, ribonucleases, and lipases that preferentially degrade the modified components of proteins, DNA, and lipids, respectively. In addition, protection against free radical damage can be afforded by the inclusion of certain vitamins (vitamin C and vitamin E), carotenoids ( -carotene), flavanoids, and other antioxidants in the diet, which inhibit the initiation of the free radical processes or can act as chain-breaking antioxidants. In spite of the development of various antioxidant systems to counteract the damaging effects of ROS, with age, the cell succumbs to oxidative stress, which has been defined as an imbalance that is shifted towards the prooxidant system relative to the antioxidant systems in the body, leading to cell damage and ultimately cell death. Oxidative stress is known to cause lipid peroxidation, protein oxidation, DNA fragmentation, impairment of cellular energy status, and disruption of ion homeostasis. Free radical–mediated oxida450 ANNALS NEW YORK ACADEMY OF SCIENCES tive stress has also been implicated in causing damage leading to the pathology of aging and such age-associated disorders as stroke, amyotrophic lateral sclerosis, Parkinson’s disease, and Alzheimer’s disease (AD). Models of aging, such as hyperoxia, have also been investigated. OXIDATIVE STRESS AND AGING The free radical theory of aging states that the progressive erosion of cellular components occurring due to free radical damage leads to aging and ultimately results in the death of the organism. Several studies report that the rate of metabolism is directly related to the rate of aging. Oxygen consumption and ROS production are closely related, and hence it is hypothesized that, in animals having high metabolic rates, the levels of ROS are also elevated due to increased oxidative stress. In vivo studies have shown that the level of oxidative stress increases during aging. It has never been conclusively established whether this increase in oxidative stress is due to decreased antioxidant levels or due to an increase in production of prooxidant molecules in the cell. Thus oxidative stress can play an important role in aging either by affecting the efficiency with which the antioxidant defenses and/or repair mechanisms operate or by causing structural and functional changes within those molecules, or it can accelerate aging by altering the gene expression of the various cellular components. Free radical oxidative stress with consequential protein oxidation and lipid peroxidation can lead to cell death. Our laboratory has been involved with factors associated with oxidative stress that alter the physical and chemical states of cortical synaptosomal membranes. Several in vivo and in vitro models of oxidative stress have been developed and studied for this purpose. The focus of this review is a summary of work done in our laboratory on changes in protein structure and function in three in vivo models of free radical–induced oxidative stress, namely, hyperoxia, ischemia–reperfusion injury (IRI), and accelerated senescence, and the protection offered by the free radical scavenger, Ntert-butyl-phenylnitrone (PBN), against these damages. This review also summarizes similar protein damage seen in an in vitro model of oxidative stress, that is, synaptosomes exposed to amyloid -peptide, a peptide implicated in the pathology of AD, and the protective effects of the antioxidant vitamin E against the ensuing damage. MARKERS OF MEMBRANE PROTEIN DAMAGE Protein Conformational Changes Alterations in protein conformations can lead to increased aggregation, fragmentation, distortion of secondary and tertiary structure, susceptibility to proteolysis, and diminution of normal function. The technique of EPR (electron paramagnetic resonance), in conjunction with proteinand lipid-specific spin labels, is used to study membrane protein and lipid conformational changes. The power of EPR spin labeling methods derives from the extreme sensitivity of EPR, the information that can be obtained about motion and polarity of the local microenvironment near the paramagnetic center of the spin label, the relatively simple spectra that need to be analyzed, that opaque samples not BUTTERFIELD et al.: STRUCTURAL AND FUNCTIONAL CHANGES 451 susceptible to light-scattering effects common to optical spectroscopy can be efficiently studied, and the fact that generally, only the spin label is paramagnetic, that is, the biological system is EPR silent and hence, does not interfere with the spectrum. The sulfhydrylselective spin label MAL-6 (2,2,6,6-tetramethyl-4-maleimidopiperidine-1-oxyl), which covalently binds only to the -SH groups on the proteins, is the predominant spin label employed. MAL-6 is a stable paramagnetic nitroxide that generates an EPR spectrum on binding membrane proteins. Depending on whether the MAL-6 binds to -SH groups deeply within clefts of the protein or close to the protein surface, the spin label is either strongly (S) or weakly (W) immobilized, respectively. This difference in spin-label motion causes both a broad and a narrow low-field line. The ratio of the spectral amplitudes of the MI low-field resonance line of the weakly immobilized site (W) to that of the strongly immobilized site (S), referred to as the W/S ratio, is a sensitive measure of changes in the physical state of the protein. Decreased W/S ratios indicate increased protein–protein interaction and decreased segmental motion and/or conformational changes in the proteins that were labeled; the converse is also the case. Earlier studies in our laboratory using other conditions of free radical–induced oxidative stress, such as hydroxyl radical generation, sepsis-associated lipopolysaccharide, or menadione have shown W/S ratios to be lowered in each case. Hence, the W/S ratio can be used as a valuable marker of protein alterations.