Pamela A. Szweda

Proteolysis, free radicals, and aging,

Aging is accompanied by declines in cellular proteolytic capacity. Proteolytic processing is an important step in numerous cellular processes required for normal metabolic function. These include regulation of protein turnover, degradation of altered forms of protein, signal transduction, protein sorting/trafficking, receptor-mediated endo- and exocytosis, stress/immune responses, and activation of gene transcription. Thus, loss of cellular proteolytic function is likely to contribute to the enhanced fragility of cells from senescent relative to young and adult organisms. Free radicals have been implicated as contributing factors to observed age-dependent declines in proteolytic capacity. The current review offers an overview of the evidence linking free radical events to functional alterations in the lysosomal system and the proteasome, two major pathways by which proteins are degraded within cells. Implications for future investigations in the field are discussed in light of these findings.

Aging, lipofuscin formation, and free radical-mediated inhibition of cellular proteolytic systems

Alterations in a wide array of physiological functions are a normal consequence of aging. Importantly, aged individuals exhibit an enhanced susceptibility to various degenerative diseases and appear less able than their young and adult counterparts to withstand (patho)physiological stress. Elucidation of mechanisms at play in the aging process would benefit the development of effective strategies for enhancing the quality of life for the elderly. It is likely that decrements in cellular and physiological function that occur during aging are the net result of numerous interacting factors. The current review focuses on the potential contribution(s) of free radical-mediated modifications to protein structure/function and alterations in the activities of two major proteolytic systems within cells, lysosomes and the proteasome, to the age-dependent accumulation of fluorescent intracellular granules, termed lipofuscin. Specifically, aging appears to influence the interplay between the occurrences of free radical-derived modifications to protein and the ability of cells to carry out critical proteolytic functions. We present immunochemical and ultrastructural evidence demonstrating the occurrence of a fluorescent protein cross-link derived from free radical-mediated reaction(s) within lipofuscin granules of rat cerebral cortex neurons. In addition, we provide evidence that a fluorophore-modified protein present in lipofuscin granules is the alpha subunit of F1F0-ATP synthase, a mitochondrial protein. It has previously been shown that protein(s) bearing this particular fluorescent cross-link are resistant to proteolysis and can inhibit the proteasome in a non-competitive fashion (J. Biol. Chem. 269 (1994a) 21639; FEBS Lett. 405 (1997) 21). Therefore, the current findings demonstrate that free radical-mediated modifications to protein(s) that lead to the production of inhibitor(s) of cellular proteolytic systems are present on specific protein components of lipofuscin. In addition, the mitochondrial origin of one of these proteins indicates specific intracellular pathways likely to be influenced by free radical events and participate in the formation of lipofuscin. The results of these studies are related to previous in vitro and in vivo observations in the field, thus shedding light on potential consequences to cellular function. In addition, future research directions suggested by the available evidence are discussed.

Selective inactivation of redox-sensitive mitochondrial enzymes during cardiac reperfusion

Reperfusion of ischemic myocardial tissue results in an increase in mitochondrial free radical production and declines in respiratory activity. The effects of ischemia and reperfusion on the activities of Krebs cycle enzymes, as well as enzymes involved in electron transport, were evaluated to provide insight into whether free radical events are likely to affect enzymatic and mitochondrial function(s). An in vivo rat model was utilized in which ischemia is induced by ligating the left anterior descending coronary artery. Reperfusion, initiated by release of the ligature, resulted in a significant decline in NADH-linked ADP-dependent mitochondrial respiration as assessed in isolated cardiac mitochondria. Assays of respiratory chain complexes revealed reduction in the activities of complex I and, to a lesser extent, complex IV exclusively during reperfusion, with no alterations in the activities of complexes II and III. Moreover, Krebs cycle enzymes alpha-ketoglutarate dehydrogenase and aconitase were susceptible to reperfusion-induced inactivation with no decline in the activities of other Krebs cycle enzymes. The decline in alpha-ketoglutarate dehydrogenase activity during reperfusion was associated with a loss in native lipoic acid on the E2 subunit, suggesting oxidative inactivation. Inhibition of complex I in vitro promotes free radical generation. alpha-Ketoglutarate dehydrogenase and aconitase are uniquely susceptible to in vitro oxidative inactivation. Thus, our results suggest a scenario in which inhibition of complex I promotes free radical production leading to oxidative inactivation of alpha-ketoglutarate dehydrogenase and aconitase.

α-Ketoglutarate dehydrogenase: A mitochondrial redox sensor

Abstract α-Ketoglutarate dehydrogenase (KGDH), a key regulatory enzyme within the Krebs cycle, is sensitive to mitochondrial redox status. Treatment of mitochondria with H2O2 results in reversible inhibition of KGDH due to glutathionylation of the cofactor, lipoic acid. Upon consumption of H2O2, glutathione is removed by glutaredoxin restoring KGDH activity. Glutathionylation appears to be enzymatically catalysed or require a unique microenvironment. This may represent an antioxidant response, diminishing the flow of electrons to the respiratory chain and protecting sulphydryl residues from oxidative damage. KGDH is, however, also susceptible to oxidative damage. 4-Hydroxy-2-nonenal (HNE), a lipid peroxidation product, reacts with lipoic acid resulting in enzyme inactivation. Evidence indicates that HNE modified lipoic acid is cleaved from KGDH, potentially the first step of a repair process. KGDH is therefore a likely redox sensor, reversibly altering metabolism to reduce oxidative damage and, under severe oxidative stress, acting as a sentinel of mitochondrial viability.

Aging: a shift from redox regulation to oxidative damage.

Proteins, nucleic acids, and lipids can undergo various forms of oxidative modification. In numerous instances, these modifications result in irreversible loss of function. The age-dependent accumulation of oxidatively modified and dysfunctional macromolecules provides the basis for the free radical theory of aging. Pro-oxidants, however, are also capable of catalyzing fully reversible modifications to protein. It is increasingly apparent that these reactions participate in redox-dependent regulation of cell metabolism and response to stress. The adventitious use of free radical species adds complexity to the experimental and theoretical manner in which the free radical theory is to be tested and considered. Elucidation of mechanisms by which reversible oxidative processes are controlled, the components involved, and the metabolic consequences and how they are altered with age will provide new insight on the aging process and attempts to delay the inevitable.

Dissociation of Cytochrome c from the Inner Mitochondrial Membrane during Cardiac Ischemia

Mitochondria isolated from ischemic cardiac tissue exhibit diminished rates of respiration and ATP synthesis. The present study was undertaken to determine whether cytochrome c release was responsible for ischemia-induced loss in mitochondrial function. Rat hearts were perfused in Langendorff fashion for 60 min (control) or for 30 min followed by 30 min of no flow ischemia. Mitochondria isolated from ischemic hearts in a buffer containing KCl exhibited depressed rates of maximum respiration and a lower cytochrome c content relative to control mitochondria. The addition of cytochrome c restored maximum rates of respiration, indicating that the release of cytochrome c is responsible for observed declines in function. However, mitochondria isolated in a mannitol/sucrose buffer exhibited no ischemia-induced loss in cytochrome c content, indicating that ischemia does not on its own cause the release of cytochrome c. Nevertheless, state 3 respiratory rates remained depressed, and cytochrome c release was enhanced when mitochondria from ischemic relative to perfused tissue were subsequently placed in a high ionic strength buffer, hypotonic solution, or detergent. Thus, events that occur during ischemia favor detachment of cytochrome c from the inner membrane increasing the pool of cytochrome c available for release. These results provide insight into the sequence of events that leads to release of cytochrome c and loss of mitochondrial respiratory activity during cardiac ischemia/reperfusion.

Regulated Production of Free Radicals by the Mitochondrial Electron Transport Chain: Cardiac Ischemic Preconditioning

Excessive production of free radicals by mitochondria is associated with, and likely contributes to, the progression of numerous pathological conditions. Nevertheless, the production of free radicals by the mitochondria may have important biological functions under normal or stressed conditions by activating or modulating redox-sensitive cellular signaling pathways. This raises the intriguing possibility that regulated mitochondrial free radical production occurs via mechanisms that are distinct from pathologies associated with oxidative damage. Indeed, the capacity of mitochondria to produce free radicals in a limited manner may play a role in ischemic preconditioning, the phenomenon whereby short bouts of ischemia protect from subsequent prolonged ischemia and reperfusion. Ischemic preconditioning can thus serve as an important model system for defining regulatory mechanisms that allow for transient, signal-inducing, production of free radicals by mitochondria. Defining how these mechanism(s) occur will provide insight into therapeutic approaches that minimize oxidative damage without altering normal cellular redox biology. The aim of this review is to present and discuss evidence for the regulated production of superoxide by the electron transport chain within the ischemic preconditioning paradigm of redox regulation.

[51] Detection of 4-hydroxy-2-nonenol adducts following lipid peroxidation from ozone exposure

Publisher Summary Peroxidation of membrane lipids results in free radical-mediated fragmentation of polyunsaturated fatty acids, giving rise to various aldehydes, alkenals, and hydroxyalkenals. Many of these products are cytotoxic and their effects are proposed to be mediated by reactivity toward specific proteins. 4-Hydroxy-2-nonenal (HNE), an α, β-unsaturated aldehyde, is a major and the most cytotoxic product of lipid peroxidation. As such, HNE is likely an important mediator of free radical damage to cells. Incubation of proteins with HNE results in enzyme inactivation followed by the formation of inter- and intramolecular protein cross-links. The HNE cross-linked protein exhibits fluorescence with spectral properties similar to pigments that accumulate during the progression of certain degenerative diseases (ceroid) and aging (lipofuscin). Furthermore, the HNE cross-linked protein is resistant to proteolysis and acts as a potent noncompetitive inhibitor of the multicatalytic proteosome. Detection of the HNE-modified protein serves as an important index of free radical/lipid peroxidation-derived damage, and characterization of specific protein targets of HNE modification may identify proteins highly susceptible to oxidative modification(s).

Neurofilaments are the major neuronal target of hydroxynonenal-mediated protein cross-links

Abstract Lipid peroxidation generates reactive aldehydes, most notably hydroxynonenal (HNE), which covalently binds amino acid residue side chains leading to protein inactivation and insolubility. Specific adducts of lipid peroxidation have been demonstrated to be intimately associated with pathological lesions of Alzheimers disease (AD), suggesting that oxidative stress is a major component in the disease. Here, we examined the HNE-cross-linking modifications by using an antibody specific for a lysine–lysine cross-link. Since in a prior study we noted no immunolabeling of neuritic plaques or neurofibrillary tangles but instead found strong labeling of axons, we focused this study on axons. Axonal labeling was examined in mouse sciatic nerve, and immunoblotting showed the cross-link was restricted to neurofilament heavy and medium subunits, which while altering migration, did not indicate larger NF aggregates, indicative of intermolecular cross-links. Examination of mice at various ages showed the extent of modification remaining relatively constant through the life span. These findings demonstrate lipid-cross-linking peroxidation primarily involves lysine-rich neurofilaments and is restricted to intramolecular cross-links.