Nuclear activation of proteasome in oxidative stress and aging

Poly(ADP-ribosyl)ation reactions are of interest in recent years and they take place in DNA repair in different processes especially following oxidative nuclear damage. Proteasomal reactions also take place in repair following oxidative nuclear damage with the degradation of oxidized histones. Antitumor chemotherapy is generally believed to act via the oxidation of nuclear material in the tumor cells. Adaptation to oxidative stress appears to be one element in the development of long-term resistance to many chemotherapeutic drugs. The 20S proteasome has been shown to be largely responsible for the degradation of oxidatively modified proteins in the nucleus. Tumor cells are supposed to have a higher nuclear proteasome activity than do nonmalignant cells. Poly(ADP-ribosyl)ation reactions take place in the tumor cells as a consequence of chemotherapy. Such a reaction might occur with the 20S proteasome ?which is known to increase the activity- and also with histones ?which is firstly shown to decrease the degradation in this study. After hydrogen peroxide treatment of HT22 cells, degradation of the model peptide substrate suc-LLVY-MCA and degradation of oxidized histones in nuclei increased accompanied by an increase in PARP-1 mRNA expression. In the recovery of the level of protein carbonyls, single strand breaks and 8-OHdG, proteasome and PARP-1 were shown to play a role together. This was tested with inhibitor treatments. The proteasomal activation following poly(ADP-ribosyl)ation of proteasome and the decrease in poly(ADP-ribosyl)ation of histones and increase in the proteasomal degradation of histones following H2O2 treatment confirmed our hypothesis. The second part of the thesis shows the changes in PARP-1 and proteasome in different aged fibroblasts with population doublings 19, 36, and 56. The nuclear protective mechanisms were shown to be effected during the senescence process. PARP-1 protein amount decreased whereas there was no change in proteasome amount. PARP activation following H2O2 treatment increased only in young and middle aged cells. In the nuclear extracts of young and old cells, poly(ADP-ribosyl)ation potentials were tested with NAD+ addition into the reaction. In addition to that active proteasome and PARP enzymes were added into the reaction and proteasome activity was measured. With active PARP, proteasome activity was increased both in young and old cells whereas there was no increase in old cells without PARP addition. These results show that proteasome activation is mainly limited by PARP activity. Taken together all results demonstrate the importance of PARP mediated proteasome activation in the repair of oxidatively damaged chromatin.Poly(ADP-ribosyl)ierungs-Reaktionen sind seit einigen Jahren im Zentrum des wissenschaftlichen Interesses. Sie finden während der DNA-Reparatur, insbesondere nach oxidativer Schädigung des Zellkerns statt. Proteasomale Reaktionen treten ebenfalls nach nukleärem Schaden auf und beinhalten den Abbau oxidierter Histone. Eine Antitumor-Chemotherapie wirkt in Tumorzellen häufig über die Oxidation nukleärem Materials. Eine Adaptation zu oxidativem Stress ist häufig ein Bestandteil der Entwicklung von Langzeit-Resistenzen gegen viele Chemotherapeutika. Es wurde häufig gezeigt, dass das 20S Proteasom für den Abbau oxidativ modifizierter Proteine im Zellkern verantwortlich ist. Tumorzellen haben eine höhere nukleäre Proteasomaktivität als nichtmaligne Zellen. Poly(ADP-ribosyl)ierungsreaktionen finden im Zellkern als eine Konsequenz der Chemotherapie statt. Solch eine Reaktion erfolgt auch mit dem 20S Proteasom ? gefolgt von einer Erhöhung der Aktivität- und auch mit Histonen, mit der Konsequenz eines reduzierten Abbau, wie in dieser Studie erstmals gezeigt. Nach einer Wasserstoffperoxidbelastung von HT22-Zellen steigt der Abbau des Modellpeptides suc-LLVY-MCA und von oxidierten Histonen im Zellkern ? begleitet von einem Anstieg der PARP-1 mRNA. Die Wiederherstellung des Spiegels der Proteincarbonyle, der Einzelstrangbrüche und des 8-OHdG setzt eine Interaktion von PARP-1 und des Proteasoms voraus. Dieses wurde mit Inhibitor-Studien untersucht. Die proteasomale Aktivierung nach Poly(ADP-ribosyl)ierung und der Abfall einer Poly(ADP-ribosyl)ierung der Histone sowie ein Anstieg des Histonabbaus nach H2O2?induziertem Stress, bestätigten diese Hypothese. Der zweite Teil der Arbeit beschäftigt sich mit der Veränderung der PARP-1 und des Proteasoms während der Fibroblastenalterung. Es wurden Zellen mit den Populationsverdopplungen 19, 36 und 56 untersucht. Die nukleären Schutzmechanismen waren vom Seneszenzprozess beeinflusst. Der PARP-1-Proteingehalt fiel, während das Proteasom konstant blieb. Wasserstoffperoxid induzierte eine PARP-Aktivität nur in jungen und mittelalten Zellen. In nukleären Extrakten von jungen und alten Zellen wurde das Poly(ADP-ribosyl)ierungs-Potential nach NAD+ Zugabe gemessen. Zusätzlich wurden aktives Proteasom und PARP hinzugegeben und die Proteasomaktivität gemessen. Nach Addition von aktiver PARP ist die proteasomale Aktivität in jungen und alten Zellen angestiegen, wobei es zu keinem Anstieg in alten Zellen ohne PARP-Addition gab. Diese Untersuchungen zeigen, dass die proteasomale Aktivierung im wesentlichen durch die PARP-Aktivierung limitiert ist. Zusammengefasst zeigen meine Resultate die Wichtigkeit der PARP-vermittelten proteasomalen Aktivierung in der Reparatur oxidativ geschädigten Chromatins

Lipid Rafts and Redox Regulation of Cellular Signaling in Cholesterol Induced Atherosclerosis

Redox mediated signaling mechanisms play crucial roles in the pathogenesis of several cardiovascular diseases. Atherosclerosis is one of the most important disorders induced mainly by hypercholesterolemia. Oxidation products and related signaling mechanisms are found within the characteristic biomarkers of atherosclerosis. Several studies have shown that redox signaling via lipid rafts play a significant role in the regulation of pathogenesis of many diseases including atherosclerosis. This review attempts to summarize redox signaling and lipid rafts in hypercholesterolemia induced atherosclerosis

Turnover of oxidatively modified proteins: the usage of in vitro and metabolic labeling

Cellular reactions to oxidative stress always include a response in the protein turnover. Therefore, cellular handling of proteins is important to observe. In this method review, radioactive labeling of proteins in vitro and in intact cells is described. The use of techniques based on the radioactive quantification of amino acids is much more selective and reliable than other nonradioactive methods for studying the protein turnover of both long- and short-lived proteins. Variations of such measurements allow one to measure protein synthesis, protein degradation, formation of insoluble proteins, and, perhaps, the turnover of individual proteins. (C) 2008 Elsevier Inc. All rights reserved

Oxidized Proteins As a Biomarker of Oxidative Stress in Aging and Diseases

Oxidative stress is an inevitable process of the aerobic life. Among the effects of oxidative stress, protein oxidation takes an important place because of the high protein abundance in the organism. The degradation of non-functional, oxidized proteins is an essential function of intra- and extracellular proteolytic systems. However, severely oxidized proteins are poor substrates for degradation and may accumulate. This process of accumulation of oxidized, cross-linked protein material is involved in the physiology and pathophysiology of aging and many diseases