Comparative resistance of the 20S and 26S proteasome to oxidative stress.
ABSTRACT: Oxidatively modified ferritin is selectively recognized and degraded by the 20S proteasome. Concentrations of hydrogen peroxide (H2O2) higher than 10 micromol/mg of protein are able to prevent proteolytic degradation. Exposure of the protease to high amounts of oxidants (H2O2, peroxynitrite and hypochlorite) inhibits the enzymic activity of the 20S proteasome towards the fluorogenic peptide succinyl-leucine-leucine-valine-tyrosine-methylcoumarylamide (Suc-LLVY-MCA), as well as the proteolytic degradation of normal and oxidant-treated ferritin. Fifty per cent inhibition of the degradation of the protein substrates was achieved using 40 micromol of H2O2/mg of proteasome. No change in the composition of the enzyme was revealed by electrophoretic analysis up to concentrations of 120 micromol of H2O2/mg of proteasome. In further experiments, it was found that the 26S proteasome, the ATP- and ubiquitin-dependent form of the proteasomal system, is much more susceptible to oxidative stress. Whereas degradation of the fluorogenic peptide, Suc-LLVY-MCA, by the 20S proteasome was inhibited by 50% with 12 micromol of H2O2/mg, 3 micromol of H2O2/mg was enough to inhibit ATP-stimulated degradation by the 26S proteasome by 50%. This loss in activity could be followed by the loss of band intensity in the non-denaturing gel. Therefore we concluded that the 20S proteasome was more resistant to oxidative stress than the ATP- and ubiquitin-dependent 26S proteasome. Furthermore, we investigated the activity of both proteases in K562 cells after H2O2 treatment. Lysates from K562 cells are able to degrade oxidized ferritin at a higher rate than non-oxidized ferritin, in an ATP-independent manner. This effect could be followed even after treatment of the cells with H2O2 up to a concentration of 2mM. The lactacystin-sensitive ATP-stimulated degradation of the fluorogenic peptide Suc-LLVY-MCA declined, after treatment of the cells with 1mM H2O2, to the same level as that obtained without ATP stimulation. Therefore, we conclude that the regulation of the 20S proteasome by various regulators takes place during oxidative stress. This provides further evidence for the role of the 20S proteasome in the secondary antioxidative defences of mammalian cells.
Project description:INTRODUCTION:In cancer treatment an attempt has been made to pharmacologically regulate the proteasome functions, thus the aim was to test whether 20S proteasome chymotrypsin-like (ChT-L) activity has a role in glial brain tumors. Furthermore, we analyzed the correlation between proteasome activity and IL-8, CCL2, NF-?B1 and NF-?B2 concentrations, which impact on brain tumors has already been indicated. METHODS:Plasma 20S proteasome ChT-L activity was assayed using the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence of SDS. IL-8, CCL2, NF-?B1 and NF-?B2 concentration was analyzed with the use of ELISA method. Immunohistochemistry for IDH1-R132H was done on 5-microns-thick formalin-fixed, paraffin-embedded tumor sections with the use of antibody specific for the mutant IDH1-R132H protein. Labelled streptavidin biotin kit was used as a detection system. RESULTS:Brain tumor patients had statistically higher 20S proteasome ChT-L activity (0.649 U/mg) compared to non-tumoral individuals (0.430 U/mg). IDH1 wild-type patients had statistically higher 20S proteasome ChT-L activity (1.025 U/mg) compared to IDH1 mutants (0.549 U/mg). 20S proteasome ChT-L activity in brain tumor patients who died as the consequence of a tumor (0.649) in the following 2 years was statistically higher compared to brain tumor patients who lived (0.430 U/mg). In brain tumor patients the 20S proteasome ChT-L activity positively correlated with IL-8 concentration. CONCLUSIONS:Elevated 20S proteasome ChT-L activity was related to the increased risk of death in glial brain tumor patients. A positive correlation between 20S proteasome ChT-L activity and IL-8 concentration may indicate the molecular mechanisms regulating glial tumor biology. Thus research on proteasomes may be important and should be carried out to verify if this protein complexes may represent a potential therapeutic target to limit brain tumor invasion.
Project description:The 20S proteasome is a self-compartmentalized protease which degrades unfolded polypeptides and has been purified from eucaryotes, gram-positive actinomycetes, and archaea. Energy-dependent complexes, such as the 19S cap of the eucaryal 26S proteasome, are assumed to be responsible for the recognition and/or unfolding of substrate proteins which are then translocated into the central chamber of the 20S proteasome and hydrolyzed to polypeptide products of 3 to 30 residues. All archaeal genomes which have been sequenced are predicted to encode proteins with up to approximately 50% identity to the six ATPase subunits of the 19S cap. In this study, one of these archaeal homologs which has been named PAN for proteasome-activating nucleotidase was characterized from the hyperthermophile Methanococcus jannaschii. In addition, the M. jannaschii 20S proteasome was purified as a 700-kDa complex by in vitro assembly of the alpha and beta subunits and has an unusually high rate of peptide and unfolded-polypeptide hydrolysis at 100 degrees C. The 550-kDa PAN complex was required for CTP- or ATP-dependent degradation of beta-casein by archaeal 20S proteasomes. A 500-kDa complex of PAN(Delta1-73), which has a deletion of residues 1 to 73 of the deduced protein and disrupts the predicted N-terminal coiled-coil, also facilitated this energy-dependent proteolysis. However, this deletion increased the types of nucleotides hydrolyzed to include not only ATP and CTP but also ITP, GTP, TTP, and UTP. The temperature optimum for nucleotide (ATP) hydrolysis was reduced from 80 degrees C for the full-length protein to 65 degrees C for PAN(Delta1-73). Both PAN protein complexes were stable in the absence of ATP and were inhibited by N-ethylmaleimide and p-chloromercuriphenyl-sulfonic acid. Kinetic analysis reveals that the PAN protein has a relatively high V(max) for ATP and CTP hydrolysis of 3.5 and 5.8 micromol of P(i) per min per mg of protein as well as a relatively low affinity for CTP and ATP with K(m) values of 307 and 497 microM compared to other proteins of the AAA family. Based on electron micrographs, PAN and PAN(Delta1-73) apparently associate with the ends of the 20S proteasome cylinder. These results suggest that the M. jannaschii as well as related archaeal 20S proteasomes require a nucleotidase complex such as PAN to mediate the energy-dependent hydrolysis of folded-substrate proteins and that the N-terminal 73 amino acid residues of PAN are not absolutely required for this reaction.
Project description:Hallmarks of aging include loss of protein homeostasis and dysregulation of stress-adaptive pathways. Loss of adaptive homeostasis, increases accumulation of DNA, protein, and lipid damage. During acute stress, the Cnc-C (Drosophila Nrf2 orthologue) transcriptionally-regulated 20S proteasome degrades damaged proteins in an ATP-independent manner. Exposure to very low, non-toxic, signaling concentrations of the redox-signaling agent hydrogen peroxide (H2O2) cause adaptive increases in the de novo expression and proteolytic activity/capacity of the 20S proteasome in female D. melanogaster (fruit-flies). Female 20S proteasome induction was accompanied by increased tolerance to a subsequent normally toxic but sub-lethal amount of H2O2, and blocking adaptive increases in proteasome expression also prevented full adaptation. We find, however, that this adaptive response is both sex- and age-dependent. Both increased proteasome expression and activity, and increased oxidative-stress resistance, in female flies, were lost with age. In contrast, male flies exhibited no H2O2 adaptation, irrespective of age. Furthermore, aging caused a generalized increase in basal 20S proteasome expression, but proteolytic activity and adaptation were both compromised. Finally, continual knockdown of Keep1 (the cytosolic inhibitor of Cnc-C) in adults resulted in older flies with greater stress resistance than their age-matched controls, but who still exhibited an age-associated loss of adaptive homeostasis.
Project description:Proteostasis is critical for the maintenance of life. In neuronal cells an imbalance between protein synthesis and degradation is thought to be involved in the pathogenesis of neurodegenerative diseases during aging. Partly, this seems to be due to a decrease in the activity of the ubiquitin-proteasome system, wherein the 20S/26S proteasome complexes catalyse the proteolytic step. We have characterised 20S and 26S proteasomes from cerebrum, cerebellum and hippocampus of 3 weeks old (young) and 24 month old (aged) rats. Our data reveal that the absolute amount of the proteasome is not dfferent between both age groups. Within the majority of standard proteasomes in brain the minute amounts of immuno-subunits are slightly increased in aged rat brain. While this goes along with a decrease in the activities of 20S and 26S proteasomes to hydrolyse synthetic fluorogenic tripeptide substrates from young to aged rats, the capacity of 26S proteasomes for degradation of poly-Ub-model substrates and its activation by poly-Ub-substrates is not impaired or even slightly increased in brain of aged rats. We conclude that these alterations in proteasome properties are important for maintaining proteostasis in the brain during an uncomplicated aging process.
Project description:The ATP-powered protein degradation machinery plays essential roles in maintaining protein homeostasis in all organisms. Robust proteolytic activities are typically sequestered within protein complexes to avoid the fatal removal of essential proteins. Because the openings of proteolytic chambers are narrow, substrate proteins must undergo unfolding. AAA superfamily proteins (ATPases associated with diverse cellular activities) are mostly located at these openings and regulate protein degradation appropriately. The 26S proteasome, comprising 20S peptidase and 19S regulatory particles, is the major ATP-powered protein degradation machinery in eukaryotes. The 19S particles are composed of six AAA proteins and 13 regulatory proteins, and bind to both ends of a barrel-shaped proteolytic chamber formed by the 20S peptidase. Several recent studies have reported that another AAA protein, Cdc48, can replace the 19S particles to form an alternative ATP-powered proteasomal complex, i.e., the Cdc48-20S proteasome. This review focuses on our current knowledge of this alternative proteasome and its possible linkage to amyotrophic lateral sclerosis.
Project description:Proteasomal degradation is altered in many disease phenotypes including cardiac hypertrophy, a prevalent condition leading to heart failure. Our recent investigations identified heterogeneous subpopulations of proteasome complexes in the heart and implicated multiple mechanisms for their regulation.The study aimed at identification of molecular mechanisms changing proteasome function in the hypertrophic heart.Proteasome function, expression, and assembly were analyzed during the development of cardiac hypertrophy induced by ?-adrenergic stimulation. The analysis revealed, for the first time, divergent regulation of proteasome function in cardiac hypertrophy. Proteasome complexes have 3 different proteolytic activities, which are ATP-dependent for 26S complexes (19S assembled with 20S) and ATP-independent for 20S core particles. The 26S activities were enhanced in hypertrophic hearts, partially because of increased expression and assembly of 19S subunits with 20S core complexes. In contrast, caspase- and trypsin-like 20S activities were significantly decreased. Activation of endogenous cAMP-dependent protein kinase (PKA) rescued the depressed 20S functions, supporting the notion that PKA signaling is a positive regulator of protein degradation in the heart. Chymotrypsin-like 20S activity was stably maintained during cardiac remodeling, indicating a switch in proteasome subpopulations, which was supported by altered expression and incorporation of inducible ? subunits.Three novel mechanisms for the regulation of proteasome activities were discovered in the development of cardiac hypertrophy: (1) increased incorporation of inducible subunits in 20S proteasomes; (2) enhanced 20S sensitivity to PKA activation; and (3) increased 26S assembly. PKA modulation of proteasome complexes may provide a novel therapeutic avenue for restoration of cardiac function in the diseased myocardium.
Project description:The proteasome catalyzes the degradation of many mis-folded proteins, which are otherwise cytotoxic. There is interest in the discovery of proteasome agonists, but previous efforts to do so have been disappointing.The cleavage of small fluorogenic peptides is used routinely as an assay to screen for proteasome modulators. We have developed follow-on assays that employ more physiologically relevant substrates.To demonstrate the efficacy of this workflow, the NIH Clinical Collection (NCC) was screened. While many compounds stimulated proteasome-mediated proteolysis of the pro-fluorogenic peptide substrates, most failed to evince activity in assays with larger peptide or protein substrates. We also show that two molecules claimed previously to be proteasome agonists, oleuropein and betulinic acid, indeed accelerate hydrolysis of the fluorogenic substrate, but have no effect on the turnover of a mis-folded protein in vitro or in cellulo. However, two small molecules from the NCC, MK-866 and AM-404, stimulate the proteasome-mediated turnover of a mis-folded protein in living cells by 3- to 4-fold.Assays that monitor the proteasome-mediated degradation of larger peptides and proteins can distinguish bona fide agonists from compounds only able to stimulate the cleavage of short, non-physiologically relevant peptides.A suite of assays has been established that allows the discovery of bona fide proteasome agonists. AM-404 and MK-866 can be useful tools for cell culture experiments, and can serve as scaffolds to generate more potent 20S stimulators.
Project description:Oxidized cytoplasmic and nuclear proteins are normally degraded by the proteasome, but accumulate with age and disease. We demonstrate the importance of various forms of the proteasome during transient (reversible) adaptation (hormesis), to oxidative stress in murine embryonic fibroblasts. Adaptation was achieved by 'pre-treatment' with very low concentrations of H2O2, and tested by measuring inducible resistance to a subsequent much higher 'challenge' dose of H2O2. Following an initial direct physical activation of pre-existing proteasomes, the 20S proteasome, immunoproteasome and PA28?? regulator all exhibited substantially increased de novo synthesis during adaptation over 24 h. Cellular capacity to degrade oxidatively damaged proteins increased with 20S proteasome, immunoproteasome and PA28?? synthesis, and was mostly blocked by the 20S proteasome, immunoproteasome and PA28 siRNA (short interfering RNA) knockdown treatments. Additionally, PA28??-knockout mutants achieved only half of the H2O2-induced adaptive increase in proteolytic capacity of wild-type controls. Direct comparison of purified 20S proteasome and immunoproteasome demonstrated that the immunoproteasome can selectively degrade oxidized proteins. Cell proliferation and DNA replication both decreased, and oxidized proteins accumulated, during high H2O2 challenge, but prior H2O2 adaptation was protective. Importantly, siRNA knockdown of the 20S proteasome, immunoproteasome or PA28?? regulator blocked 50-100% of these adaptive increases in cell division and DNA replication, and immunoproteasome knockdown largely abolished protection against protein oxidation.
Project description:Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions.
Project description:H2O2-induced programmed cell death (PCD) of tobacco Bright Yellow-2 (BY-2) cells is mediated by reactive carbonyl species (RCS), degradation products of lipid peroxides, which activate caspase-3-like protease (C3LP). Here, we investigated the mechanism of RCS accumulation in the H2O2-induced PCD of BY-2 cells. The following biochemical changes were observed in 10-min response to a lethal dose (1.0 mM) of H2O2, but they did not occur in a sublethal dose (0.5 mM) of H2O2. (1) The C3LP activity was increased twofold. (2) The intracellular levels of RCS, i.e., 4-hydroxy-(E)-hexenal and 4-hydroxy-(E)-nonenal (HNE), were increased 1.2-1.5-fold. (3) The activity of a reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent carbonyl reductase, scavenging HNE, and n-hexanal was decreased. Specifically, these are the earliest events leading to PCD. The proteasome inhibitor MG132 suppressed the H2O2-induced PCD, indicating that the C3LP activity of the 1 subunit of the 20S proteasome was responsible for PCD. The addition of H2O2 to cell-free protein extract inactivated the carbonyl reductase. Taken together, these results suggest a PCD-triggering mechanism in which H2O2 first inactivates a carbonyl reductase(s), allowing RCS levels to rise, and eventually leads to the activation of the C3LP activity of 20S proteasome. The carbonyl reductase thus acts as an ROS sensor for triggering PCD.