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T Grune

Publications and source records attributed to T Grune.

At least 37 records · Page 2Linked to original sources
PubMed2001

Lipofuscin accumulation in proliferating fibroblasts in vitro: an indicator of oxidative stress.

The amount of the ageing pigment, lipofuscin, found in replicating cells depends both on its rate of formation as well as its rate of dissolution by cell division. We present a model which allows the calculation of the lipofuscin accumulation rate from measurements of its amount and of the cell cycle duration. In two human fibroblast strains, the accumulation rate correlates well with differences in oxidative stress/antioxidative defence as measured by intracellular peroxide generation, protein carbonyl content, telomere shortening rate and replicative life span. The lipofuscin content increases with replicative age in both cultures. The rather steep increase in presenescent fibroblasts is not solely due to a slowing down of the cell turnover, but is partially caused by an increased rate of lipofuscin formation/ accumulation. This might indicate an increased level of oxidative stress in presenescent fibroblasts, or a decreased efficiency of proteolytic systems, or both. The results are in accordance with data demonstrating an adverse effect of lipofuscin accumulation on cellular protein turnover and suggest an active role for lipofuscin accumulation in cellular senescence.

Biomarkers↗
PubMed2001

The proteasome and its function in the ageing process.

The ageing process is characterized by a progressive loss of function and a decline in the functional capacities of the organism, leading to death. The nature of the processes involved in loss of functions is not well understood. A number of theories have been proposed, including a hypothesis that emphasizes the role of reactive oxygen species as a fundamental causal factor in the ageing process; among other things, oxidative damage to proteins through reactive oxygen species plays a key role in the ageing process. Oxidative modification of proteins generally causes them to become dysfunctional, and normally to undergo preferential degradation. Within the cell the main proteolytic machinery involved in the degradation of oxidized proteins is the proteasomal system, consisting of a multicatalytic protease complex--the proteasome--and numerous regulatory factors. The proteasome is a highly conserved structure that is distributed in the cytosol, nucleus and endoplasmatic reticulum of mammalian cells. As the proteasome itself is also exposed to oxidative stress during the ageing process several studies were carried out to investigate the role and the activity of the proteasomal system during ageing. This review will describe current knowledge of the activity of the protesomal system and its possible involvement in the ageing process.

Aging↗
PubMed2001

Age-related changes in protein oxidation and proteolysis in mammalian cells.

Reactive oxygen species generated as by-products of oxidative metabolism, or from environmental sources, frequently damage cellular macromolecules. Proteins are recognized as major targets of oxidative modification, and the accumulation of oxidized proteins is a characteristic feature of aging cells. An increase in the amount of oxidized proteins has been reported in many experimental aging models, as measured by the level of intracellular protein carbonyls or dityrosine, or by the accumulation of protein-containing pigments such as lipofuscin and ceroid bodies. In younger individuals, moderately oxidized soluble cell proteins appear to be selectively recognized and rapidly degraded by the proteasome. An age-related accumulation of oxidized proteins could, therefore, be a result of declining activity of the proteasome. Previous research to investigate the notion of an age-related decline in the content and/or activity of the proteasome has generated contradictory results. The latest evidence, including our own recent findings, indicates that proteasome activity does, indeed, decline during aging as the enzyme complex is progressively inhibited by oxidized and cross-linked protein aggregates. We propose that cellular aging involves both an increase in (mitochondrial) oxidant production and a progressive decline in proteasome activity. Eventually so much proteasome is inactivated that oxidized proteins begin to accumulate rapidly and contribute to cellular dysfunction and senescence.

Animals↗
PubMed2001

Proteolysis of oxidized proteins after oxygen-glucose deprivation in rat cortical neurons is mediated by the proteasome.

Oxidative injury contributes to cellular damage during and after cerebral ischemia. However, the downstream catabolic pathways of damaged cellular components in neurons are largely unknown. In the current study, the authors examined the formation of oxidized proteins and their active degradation by the proteasome. In near-pure rat primary cortical neurons, it was found that protein-bound carbonyls as markers for oxidized proteins are increased after oxygen-glucose deprivation (OGD). During and after OGD, degradation of proteins metabolically radiolabeled before OGD increases two-to threefold compared with the normal protein turnover. Proteolysis after reoxygenation was attenuated by the presence of dimethylthiourea, a radical scavenger, and was blocked by lactacystin, a specific proteasome inhibitor. Lactacystin also increased the amount of protein carbonyls formed. In contrast, the activity of the proteasome complex itself after OGD was not different from sham-washed controls. The authors suggest that oxygen-glucose deprivation increases free radicals, which, in turn, oxidize proteins that are recognized and actively degraded by the proteasome complex. This protease itself is relatively resistant against oxidative injury. The authors conclude that the proteasome may be an active part of the cellular defense system against oxidative stress after cerebral ischemia.

Acetylcysteine↗
PubMed2000

Hydrogen peroxide-induced structural alterations of RNAse A.

Proteins exposed to oxidative stress are degraded via proteolytic pathways. In the present study, we undertook a series of in vitro experiments to establish a correlation between the structural changes induced by mild oxidation of the model protein RNase A and the proteolytic rate found upon exposure of the modified protein toward the isolated 20 S proteasome. Fourier transform infrared spectroscopy was used as a structure-sensitive probe. We report here strong experimental evidence for oxidation-induced conformational rearrangements of the model protein RNase A and, at the same time, for covalent modifications of amino acid side chains. Oxidation-related conformational changes, induced by H(2)O(2) exposure of the protein may be monitored in the amide I region, which is sensitive to changes in protein secondary structure. A comparison of the time- and H(2)O(2) concentration-dependent changes in the amide I region demonstrates a high degree of similarity to spectral alterations typical for temperature-induced unfolding of RNase A. In addition, spectral parameters of amino acid side chain marker bands (Tyr, Asp) revealed evidence for covalent modifications. Proteasome digestion measurements on oxidized RNase A revealed a specific time and H(2)O(2) concentration dependence; at low initial concentration of the oxidant, the RNase A turnover rate increases with incubation time and concentration. Based on these experimental findings, a correlation between structural alterations detected upon RNase A oxidation and proteolytic rates of RNase A is established, and possible mechanisms of the proteasome recognition process of oxidatively damaged proteins are discussed.

Hydrogen Peroxide↗
PubMed2000

Differential impairment of 20S and 26S proteasome activities in human hematopoietic K562 cells during oxidative stress.

The 20S proteasome and the 26S proteasome are major components of the cytosolic and nuclear proteasomal proteolytic systems. Since proteins are known to be highly susceptible targets for reactive oxygen species, the effect of H(2)O(2) treatment of K562 human hematopoietic cells toward the activities of 20S and 26S proteasomes was investigated. While the ATP-independent degradation of the fluorogenic peptide suc-LLVY-MCA was not affected by H(2)O(2) concentrations of up to 5 mM, the ATP-stimulated degradation of suc-LLVY-MCA by the 26S proteasome began to decline at 400 microM and was completely abolished at 1 mM oxidant treatment. A combination of nondenaturing electrophoresis and Western blotting let us believe that the high oxidant susceptibility of the 26S proteasome is due to oxidation of essential amino acids in the proteasome activator PA 700 which mediates the ATP-dependent proteolysis of the 26S-proteasome. The activity of the 26S-proteasome could be recovered within 24 h after exposure of cells to 1 mM H(2)O(2) but not after 2 mM H(2)O(2). In view of the specific functions of the 26S proteasome in cell cycle control and other important physiological functions, the consequences of the higher susceptibility of this protease toward oxidative stress needs to be considered.

Acetylcysteine↗
PubMed2000

Hydrogen peroxide-mediated protein oxidation in young and old human MRC-5 fibroblasts.

It is suggested that the aging process is dependent on the action of free radicals. One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The present investigation was undertaken to reveal the proliferation-related changes in the protein oxidation and proteasome activity during and after an acute oxidative stress. It could be demonstrated that the activity of the cytosolic proteasomal system declines during proliferative senescence of human MRC-5 fibroblasts and is not able to remove oxidized proteins in old cells efficiently. Whereas in young cells removal of oxidized proteins was accompanied by an increase in the overall protein turnover, this increase in protein turnover could not be seen in old MRC-5 fibroblasts. Therefore, our studies demonstrate that old fibroblasts are much more vulnerable to the accumulation of oxidized proteins after oxidative stress and are not able to remove these oxidized proteins as efficiently as young fibroblasts.

Aldehydes↗
PubMed2000

Protein oxidation and degradation during proliferative senescence of human MRC-5 fibroblasts.

One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The aging process on a cellular level can be treated either as the ongoing proliferation until a certain number of cell divisions is reached (the Hayflick limit) or as the aging of nondividing cells, that is, the age-related changes in cells without proliferation. The present investigation was undertaken to reveal the changes in protein turnover, proteasome activity, and protein oxidation status during proliferative senescence. We were able to demonstrate that the activity of the cytosolic proteasomal system declines dramatically during the proliferative senescence of human MRC-5 fibroblasts. Regardless of the loss in activity, it could be demonstrated that there are no changes in the transcription and translation of proteasomal subunits. This decline in proteasome activity was accompanied by an increased concentration of oxidized proteins. Cells at higher proliferation stages were no longer able to respond with increased degradation of endogenous [(35)S]-Met-radiolabeled proteins after hydrogen peroxide- or quinone-induced oxidative stress. It could be demonstrated that oxidized proteins in senescent human MRC-5 fibroblasts are not as quickly removed as they are in young cells. Therefore, our study demonstrates that the accumulation of oxidized proteins and decline in protein turnover and activity of the proteasomal system are not only a process of postmitotic aging but also occur during proliferative senescence and result in an increased half-life of oxidized proteins.

Cell Division↗
PubMed2000

Reduction of plasma catecholamines in humans during clinically controlled severe underfeeding.

BACKGROUND: Sympathetic hyperactivity is one factor for alterations encountered in the plurimetabolic syndrome, a cluster of metabolic abnormalities including obesity, hyperlipidemia, sometimes hyperglycaemia, and hypertonia. It was interesting to know if prolonged severe underfeeding (230 kcal/day) leads to decreases in catecholamines in those patients. METHODS: The plasma concentrations of catecholamines in patients (n = 16) suffering from plurimetabolic syndrome were studied before and during a 16-day period of medically controlled severe underfeeding (230 kcal/day) at rest and in response to exercise. RESULTS: During the period of underfeeding, mean norepinephrine concentrations decreased at rest from 1.45 to 0. 96 nmol/liter, and in response to exercise, from 6.1 to 3.2 nmol/liter. Epinephrine concentrations decreased from 0.15 to 0.1 nmol/liter and from 0.26 to 0.17 nmol/liter, respectively. A significant decrease in catecholamine concentrations was observed only after 16 days of underfeeding. CONCLUSIONS: Clinically controlled underfeeding of patients with plurimetabolic syndrome may result in beneficial clinical and biochemical effects. The findings indicate that relatively long periods of underfeeding induce decreases in plasma catecholamine concentrations. Nevertheless, most of the fall in mean values in norepinephrine and also of the fall in blood pressure values occurred by Day 2. From those tendencies and from the significant changes in both parameters at Day 16 of severe underfeeding one could conclude that altered sympathetic nervous system activity could contribute to the fall in blood pressure.

Adult↗
PubMed2000

4-Hydroxynonenal-modified amyloid-beta peptide inhibits the proteasome: possible importance in Alzheimer's disease.

The amyloid beta-peptide (Abeta) is a 4-kDa species derived from the amyloid precursor protein, which accumulates in the brains of patients with Alzheimer's disease. Although we lack full understanding of the etiology and pathogenesis of selective neuron death, considerable data do imply roles for both the toxic Abeta and increased oxidative stress. Another significant observation is the accumulation of abnormal, ubiquitin-conjugated proteins in affected neurons, suggesting dysfunction of the proteasome proteolytic system in these cells. Recent reports have indicated that Abeta can bind and inhibit the proteasome, the major cytoslic protease for degrading damaged and ubiquitin-conjugated proteins. Earlier results from our laboratory showed that moderately oxidized proteins are preferentially recognized and degraded by the proteasome; however, severely oxidized proteins cannot be easily degraded and, instead, inhibit the proteasome. We hypothesized that oxidatively modified Abeta might have a stronger (or weaker) inhibitory effect on the proteasome than does native Abeta. We therefore also investigated the proteasome inhibitory action of Abeta1-40 (a peptide comprising the first 40 residues of Abeta) modified by the intracellular oxidant hydrogen peroxide, and by the lipid peroxidation product 4-hydroxynonenal (HNE). H2O2 modification of Abeta1-40 generates a progressively poorer inhibitor of the purified human 20S proteasome. In contrast, HNE modification of Abeta1-40 generates a progressively more selective and efficient inhibitor of the degradation of fluorogenic peptides and oxidized protein substrates by human 20S proteasome. This interaction may contribute to certain pathological manifestations of Alzheimer's disease.

Aldehydes↗
PubMed2000

Proteolysis of oxidised proteins and cellular senescence.

Aerobe living is consequently connected with a permanent oxidation of cellular proteins. Free radicals and other oxidants damage the normal intracellular protein pool. Therefore, the prevention of accumulation of oxidised cellular proteins is one of the major functions of the proteolytic machinery of mammalian cells. It is known that the multicatalytic proteinase complex, the proteasome, is the major protease that is able to recognise and degrade oxidised proteins. Cellular models are most useful to investigate biochemical changes of protein catabolism during senescence. Unfortunately, little is known about the protein turnover and the regulation of the proteasomal system as well as under oxidative stress conditions as during senescence. The proteasomal regulation during oxidative stress, protein oxidation and the changes of these processes during the ageing process are highlighted in this review.

Animals↗
PubMed2000

Oxidative stress, aging and the proteasomal system.

Oxygen free radicals and other oxidants are causing potential danger for intracellular proteins during the lifetime of cells and organisms. Therefore, protein oxidation is one of the natural consequences of aerobic life. The degradation of non-functional, oxidized proteins is an essential part of the antioxidant defenses of cells. The major proteolytic system responsible for the removal of oxidized cytosolic proteins is the proteasomal system. This system consists of the 20S 'core' proteasome and a multitude of various regulators. It is known that certain components of this system are regulated during oxidative stress and aging. One of the highlights of age-related changes of cellular metabolism is the accumulation of oxidized proteins. The question whether the accumulation of oxidized proteins during aging is due to a malfunction of the intracellular proteolytic machinery of the cell remains still unsolved. The information available about an age-related decline of the proteasomal system is very contradictory. The current literature about the proteasomal system, the regulation of this system during oxidative stress and the age-related changes are in the focus of this review.

Aging↗
PubMed2000

Ferritin oxidation in vitro: implication of iron release and degradation by the 20S proteasome.

Ferritin, the major iron storage protein in mammalian cells, was treated with various concentrations of different oxidants: xanthine/xanthine oxidase, Sin-1 (3-morpholinosydnonimine, purchased from Alexis, Grunberg, Germany), DEA-NO (Diethylamine NONOate, purchased from Calblochem-Novabiochem, Schwalbach, Germany), and hydrogen peroxide. The proteolytic susceptibility towards the isolated 20S proteasome of untreated ferritin and oxidized ferritin was measured in parallel with the iron liberated by these oxidants. With increasing hydrogen peroxide, Sin-1, and xanthine oxidase concentrations, the measured proteasomal degradation of ferritin also increased. At higher oxidant concentrations, however, the proteolytic susceptibility began to decrease. The oxidation of ferritin by DEA-NO was accompanied by a lesser increase of proteolytic susceptibility in comparison with the effects of the other oxidants. Addition of DEA-NO to Sin-1 suppressed the increase in proteolytic susceptibility of ferritin, whereas adding xanthine/xanthine oxidase had no additional effect. Iron was liberated readily from ferritin as a result of the oxidation process, although the increase in proteolytic susceptibility was not always correlated to the iron release. In fact, the degradation of oxidatively damaged ferritin was not accompanied by a further increase of free iron. Therefore, we conclude that the proteasome is a secondary antioxidative defense system that degrades only nonfunctional ferritin.

Cysteine Endopeptidases↗
PubMed2000

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-glutamyl-hydrolyzing 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).

Cell Division↗
PubMed2000

Protein oxidation and degradation during cellular senescence of human BJ fibroblasts: part II--aging of nondividing cells.

Oxidized/cross-linked intracellular protein materials, known as ceroid pigment, age pigment, or lipofuscin, accumulate in postmitotic tissues. It is unclear, however, whether diminishing proteolytic capacities play a role in the accumulation of such oxidized intracellular proteins. Previous studies revealed that the proteasome is responsible for the degradation of most oxidized soluble cytoplasmic and nuclear proteins and, we propose, for the prevention of such damage accumulations. The present investigation was undertaken to test the changes in protein turnover, proteasome activity, lysosome activity, and protein oxidation status during the aging of nondividing cells. Since the companion paper shows that both proteasome activity and the overall protein turnover decline during proliferative senescence whereas the accumulation of oxidized proteins increases significantly, we decided to use the same human BJ fibroblasts, this time at confluency, at different PD levels (including those that are essentially postmitotic) to investigate the same parameters under conditions where cells do not divide. We find that the activity of the cytosolic proteasome declines dramatically during senescence of nondividing BJ fibroblasts. The peptidyl-glutamyl-hydrolyzing activity was particularly affected. This decline in proteasome activity was accompanied by a decrease in the overall turnover of short-lived (radiolabeled) proteins in the nondividing BJ fibroblasts. On the other hand, no decrease in the actual cellular proteasome content, as judged by immunoblots, was found. The decline in the activity of the proteasome was also accompanied by an increased accumulation of oxidized proteins, especially of oxidized and cross-linked material. Unlike the loss of lysosomal function seen in our accompanying studies of proliferative senescence (1), however, the present study of hyperoxic senescence in nondividing cells actually revealed marked increases in lysosomal cathepsin activity in all but the very 'oldest' postmitotic cells. Our comparative studies of proliferating (1) and nonproliferating (this paper) human BJ fibroblasts reveal a good correlation between the accumulation of oxidized/cross-linked proteins and the decline in proteasome activity and overall cellular protein turnover during in vitro senescence, which may predict a causal relationship during actual cellular aging.

Cellular Senescence↗
PubMed2000

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 [(35)S]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.

Cathepsins↗