Tilman Grune

Degradation of oxidized proteins in mammalian cells.

Protein oxidation in vivo is a natural consequence of aerobic life. Oxygen radicals and other activated oxygen species generated as by‐products of cellular metabolism or from environmental sources cause modifications to the amino acids of proteins that generally result in loss of protein function/enzymatic activity. Oxidatively modified proteins can undergo direct chemical fragmentation or can form large aggregates due to covalent cross‐linking reactions and increased surface hydrophobicity. Mammalian cells exhibit only limited direct repair mechanisms and most oxidized proteins undergo selective proteolysis. The proteasome appears to be largely responsible for the degradation of soluble intracellular proteins. In most cells, oxidized proteins are cleaved in an ATP‐and ubiquitin‐independent pathway by the 20 S “core” proteasome. The proteasome complex recognizes hydrophobic amino acid residues, aromatic residues, and bulky aliphatic residues that are exposed during the oxidative rearrangement of secondary and tertiary protein structure: increased surface hydrophobicity is a feature common to all oxidized proteins so far tested. The recognition of such (normally shielded) hydrophobic residues is the suggested mechanism by which proteasome catalyzes the selective removal of oxidatively modified cell proteins. By minimizing protein aggregation and cross‐linking and by removing potentially toxic protein fragments, proteasome plays a key role in the overall antioxidant defenses that minimize the ravages of aging and disease.—Grune, T., Reinheckel, T., Davies, K. J. A. Degradation of oxidized proteins in mammalian cells. FASEB J. 11, 526–534 (1997)

Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease

Alzheimers disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFTs) formed of tau‐based paired helical filaments (PHFs) and extracellular β‐amyloid plaques. The degree of Alzheimer dementia correlates with the severity of PHFs and NFTs. As an intraneuronal accumulation of oxidatively damaged proteins has been found in the brains of patients with AD, a dysfunction of the proteasomal system, which degrades damaged proteins, has been assumed to cause protein aggregation and therefore neurodegeneration in AD. In this study, we revealed that such proteasome dysfunction in AD brain results from the inhibitory binding of PHF‐tau to proteasomes. We analysed the proteasome activity in brains from patients with AD and age‐matched controls, and observed a significant decrease to 56% of the control level in the straight gyrus of patients with AD. This loss of activity was not associated with a decrease in the proteasome protein. PHF‐tau co‐precipitated during proteasome immunoprecipitation and proteasome subunits could be co‐isolated during isolation of PHFs from AD brain. Furthermore, the proteasome activity in human brains strongly correlated with the amount of co‐precipitated PHF‐tau during immunoprecipitation of proteasome. Incubation of isolated proteasomes with PHF‐tau isolated from AD brain, and with PHFs after in vitro assembly from human recombinant tau protein, resulted in a distinct inhibition of proteasome activity by PHF‐tau. As this inhibition of proteasome activity was sufficient to induce neuronal degeneration and death, we suggest that PHF‐tau is able directly to induce neuronal damage in the AD brain.

Role of advanced glycation end products in cellular signaling

Improvements in health care and lifestyle have led to an elevated lifespan and increased focus on age-associated diseases, such as neurodegeneration, cardiovascular disease, frailty and arteriosclerosis. In all these chronic diseases protein, lipid or nucleic acid modifications are involved, including cross-linked and non-degradable aggregates, such as advanced glycation end products (AGEs). Formation of endogenous or uptake of dietary AGEs can lead to further protein modifications and activation of several inflammatory signaling pathways. This review will give an overview of the most prominent AGE-mediated signaling cascades, AGE receptor interactions, prevention of AGE formation and the impact of AGEs during pathophysiological processes.

Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts

We have studied the effects of hyperoxia and of cell loading with artificial lipofuscin or ceroid pigment on the postmitotic aging of human lung fibroblast cell cultures. Normobaric hyperoxia (40% oxygen) caused an irreversible senescence‐like growth arrest after about 4 wk and shortened postmitotic life span from 1–1/2 years down to 3 months. During the first 8 wk of hyperoxia‐induced ‘aging’, overall protein degradation (breakdown of [35S]methionine metabolically radiolabeled cell proteins) increased somewhat, but by 12 wk and thereafter overall proteolysis was significantly depressed. In contrast, protein synthesis rates were unaffected by 12 wk of hyperoxia. Lysosomal cathepsin‐specific activity (using the fluorogenic substrate z‐FR‐MCA) and cytoplasmic proteasome‐specific activity (measured with suc‐LLVY‐MCA) both declined by 80% or more over 12 wk. Hyperoxia also caused a remarkable increase in lipofuscin/ceroid formation and accumulation over 12 wk, as judged by both fluorescence measurements and FACscan methods. To test whether the association between lipofuscin/ceroid accumulation and decreased proteolysis might be causal, we next exposed cells to lipofuscin/ceroid loading under normoxic conditions. Lipofuscin/ceroid‐loaded cells indeed exhibited a gradual decrease in overall protein degradation over 4 wk of treatment, whereas protein synthesis was unaffected. Proteasome specific activity decreased by 25% over this period, which is important since proteasome is normally responsible for degrading oxidized cell proteins. In contrast, an apparent increase in lysosomal cathepsin activity was actually caused by a large increase in the number of lysosomes per cell. To test whether lipofuscin/ceroid could in fact directly inhibit proteasome activity, thus causing oxidized proteins to accumulate, we incubated purified proteasome with lipofuscin/ceroid preparations in vitro. We found that proteasome is directly inhibited by lipofuscin/ceroid. Our results indicate that an accumulation of oxidized proteins (and lipids) such as lipofuscin/ceroid may actually cause further increases in damage accumulation during aging by inhibiting the proteasome.–Sitte, N., Huber, M., Grune, T., Ladhoff, A., Doecke, W.‐D., von Zglinicki, T., Davies, K. J. A. Proteasome inhibition by lipofuscin/ceroid during postmitotic aging of fibroblasts. FASEB J. 14, 1490–1498 (2000)

Pathological aspects of lipid peroxidation.

Abstract Lipid peroxidation (LPO) product accumulation in human tissues is a major cause of tissular and cellular dysfunction that plays a major role in ageing and most age-related and oxidative stress-related diseases. The current evidence for the implication of LPO in pathological processes is discussed in this review. New data and literature review are provided evaluating the role of LPO in the pathophysiology of ageing and classically oxidative stress-linked diseases, such as neurodegenerative diseases, diabetes and atherosclerosis (the main cause of cardiovascular complications). Striking evidences implicating LPO in foetal vascular dysfunction occurring in pre-eclampsia, in renal and liver diseases, as well as their role as cause and consequence to cancer development are addressed.

Peroxynitrite Increases the Degradation of Aconitase and Other Cellular Proteins by Proteasome

We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite.S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50–60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I—effects of proliferative senescence

Oxidized and cross‐linked proteins tend to accumulate in aging cells. Declining activity of proteolytic enzymes, particularly the proteasome, has been proposed as a possible explanation for this phenomenon, and direct inhibition of the proteasome by oxidized and cross‐linked proteins has been demonstrated in vitro. We have further examined this hypothesis during both proliferative senescence (this paper) and postmitotic senescence (see the accompanying paper, ref 1) of human BJ fibroblasts. During proliferative senescence, we found a marked decline in all proteasome activities (trypsin‐like activity, chymotrypsin‐like activity, and peptidyl‐glutamylhydrolyzing activity) and in lysosomal cathepsin activity. Despite the loss of proteasome activity, there was no concomitant change in cellular levels of actual proteasome protein (immunoassays) or in the steady‐state levels of mRNAs for essential proteasome subunits. The decline in proteasome activities and lysosomal cathepsin activities was accompanied by dramatic increases in the accumulation of oxidized and cross‐linked proteins. Furthermore, as proliferation stage increased, cells exhibited a decreasing ability to degrade the oxidatively damaged proteins generated by an acute, experimentally applied oxidative stress. Thus, oxidized and cross linked proteins accumulated rapidly in cells of higher proliferation stages. Our data are consistent with the hypothesis that proteasome is progressively inhibited by small accumulations of oxidized and cross‐linked proteins during proliferative senescence until late proliferation stages, when so much proteasome activity has been lost that oxidized proteins accumulate at ever‐increasing rates. Lysosomes attempt to deal with the accumulating oxidized and cross‐linked proteins, but declining lysosomal cathepsin activity apparently limits their effectiveness. This hypothesis, which may explain the progressive intracellular accumulation of oxidized and cross linked proteins in aging, is further explored during postmitotic senescence in the accompanying paper (1).—Sitte, N., Merker, K., von Zglinicki, T., Grune, T., Davies, K. J. A. Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part I—effects of proliferative senescence. FASEB J. 14, 2495–2502 (2000)

Advanced Glycation End Products and Oxidative Stress in Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is a very complex and multifactorial metabolic disease characterized by insulin resistance and β cell failure leading to elevated blood glucose levels. Hyperglycemia is suggested to be the main cause of diabetic complications, which not only decrease life quality and expectancy, but are also becoming a problem regarding the financial burden for health care systems. Therefore, and to counteract the continually increasing prevalence of diabetes, understanding the pathogenesis, the main risk factors, and the underlying molecular mechanisms may establish a basis for prevention and therapy. In this regard, research was performed revealing further evidence that oxidative stress has an important role in hyperglycemia-induced tissue injury as well as in early events relevant for the development of T2DM. The formation of advanced glycation end products (AGEs), a group of modified proteins and/or lipids with damaging potential, is one contributing factor. On the one hand it has been reported that AGEs increase reactive oxygen species formation and impair antioxidant systems, on the other hand the formation of some AGEs is induced per se under oxidative conditions. Thus, AGEs contribute at least partly to chronic stress conditions in diabetes. As AGEs are not only formed endogenously, but also derive from exogenous sources, i.e., food, they have been assumed as risk factors for T2DM. However, the role of AGEs in the pathogenesis of T2DM and diabetic complications—if they are causal or simply an effect—is only partly understood. This review will highlight the involvement of AGEs in the development and progression of T2DM and their role in diabetic complications.

Age-associated analysis of oxidative stress parameters in human plasma and erythrocytes

Oxidative damage accumulation in macromolecules has been considered as a cause of cellular damage and pathology. Rarely, the oxidative stress parameters in healthy humans related to the individual age have been reported. The purpose of this study was to examine the redox status in plasma and erythrocytes of healthy individuals and determine correlations between these parameters and the aging process. The following parameters were used: malondialdehyde (MDA), protein carbonyls (PCO), 4-hydroxy-2,3-trans-nonenal (HNE), reduced glutathione (GSH), glutathione disulfide (GSSG) and uric acid (UA) in blood and plasma samples of 194 healthy women and men of ages ranging from 18 to 84 years. The results indicate that the balance of oxidant and antioxidant systems in plasma shifts in favor of accelerated oxidation during ageing. That is demonstrated by increases of MDA, HNE, GSSG and by the slight decrease of erythrocytic GSH with age. As the content of UA is more determined by metabolic and nutritional influences than by the balance between prooxidants and antioxidants there was no significant age-related change observed. For plasma concentrations of HNE the first time age-dependent reference values for healthy humans are presented.

Proteasome‐dependent degradation of oxidized proteins in MRC‐5 fibroblasts

Fibroblasts were exposed to various concentrations of hydrogen peroxide and the removal of oxidized proteins was followed by determining protein‐bound carbonyls. Fibroblasts are able to increase the turnover of metabolically radiolabeled proteins after treatment with hydrogen peroxide. It was demonstrated for the first time, that the increased protein turnover was accompanied by a removal of protein‐bound carbonyl groups. The proteasome‐specific inhibitor lactacystin was able to inhibit the elimination of protein‐bound carbonyl groups. Therefore, the key role of the proteasome in the degradation of oxidized proteins in fibroblasts could be demonstrated.