Peptidase activities of the 20/26S proteasome and a novel protease in human brain

Many neurodegenerative diseases are characterized by ubiquitin-positive protein aggregates or inclusion bodies. Ubiquitin-conjugated proteins are degraded by the 20/26S proteasome, and reduced proteasome peptidase activities in brain homogenates have been reported in pathologic lesions of Parkinson's and Alzheimer's diseases. However, it is unknown whether crude extracts of human brain contain other proteases having peptidase activities. We found a novel protease of molecular weight of approximately 105 kDa in normal human brain, which exhibited trypsin-like (T-L) and chymotrypsin-like (ChT-L) activities (corresponding to 52% and 21% of the total activities in crude extracts) but not peptidyl glutamyl peptide hydrolase activity. Both T-L and ChT-L activities of this protease were partially inhibited by proteasome inhibitors (MG132, lactacystin) and, in contrast to those of the proteasome, also by sodium dodecyl sulfate. A simple method to obtain a brain fraction specific to the 20/26S proteasome was developed. Our human brain data suggest that T-L and ChT-L activity levels of the proteasome reported previously may include those of the 105 kDa protease, an enzyme of as yet unknown biological significance, and that it is necessary to separate the proteasome from this protease to evaluate the actual status of the ubiquitin-proteasome system in neurodegenerative disorders.     

Distribution of proteasomes and of the five proteolytic activities in rat tissues

Five peptidase activities (ChT-L, T-L, PGPH, BrAAP, and SNAAP) of the proteasome, and its caseinolytic activity, were measured in crude extracts of 10 rat tissues under experimental conditions simulating those found in vivo, thereby eliminating the alterations observed with the purified enzyme. The total and individual peptidase activities varied considerably from one tissue to another, whereas the proteolytic activity measured with [(14)C]methylcasein varied no more than twofold. The tissue-specific variations in individual peptidase activities may reflect tissue-specific differences in proteasome subunit composition, or the presence of regulators. Immunological assay using an antibody directed against the iota (alpha1) subunit showed that there was no correlation between protein abundance and peptidase activity. The results also show that the different peptidase activities are not representative of proteasome distribution in the different tissues.     

Changes in 20S proteasome activity during ageing of the LOU rat

Muscular functions decline and muscle mass decreases during ageing. In the rat, there is a 27% decrease in muscle protein between 18 and 34 months of age. We examined age-related changes in the proteasome-dependent proteolytic pathway in rats at 4, 18, 24, 29 and 34 months of age. The three best characterised activities of the proteasome (chymotrypsin-like, trypsin-like and peptidylglutamyl peptide hydrolase) increased to 29 months and then decreased in the senescent animal. These variations in activity were accompanied by an identical change in the quantity of 20S proteasome measured by Western blot, whereas the S4 subunit of the 19S regulator and the quantity of ubiquitin-linked proteins remained constant. mRNA of subunits C3, C5, C9, and S4 increased in the senescent animal, but ubiquitin mRNA levels were unchanged. These findings suggest that the 20S proteasome may be partly responsible for the muscular atrophy observed during ageing in the rat.     

Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice

Premature ageing, one of the characteristics of Down syndrome (DS), may involve oxidative stress and impairment of proteasome activity. Transgenic mice overexpressing the human copper/zinc superoxide dismutase (SOD1) gene are one of the first murine models for DS and it has been shown that SOD1 overexpression might be either deleterious or beneficial. Here, we show a reduction in proteasome activities in the cortex of SOD1 transgenic mice and an associated increase in the content of oxidized SOD1 protein. As we demonstrate that in vitro oxidized SOD can inhibit purified proteasome peptidase activities, modified SOD1 might be partially responsible for proteasome inhibition shown in SOD1 transgenic mice.     

Increased muscle proteasome activities in rats fed a polyunsaturated fatty acid supplemented diet

Changes in the proteasome system, a dominant actor in protein degradation in eukaryotic cells, have been documented in a large number of physiological and pathological conditions. We investigated the influence of monounsaturated or polyunsaturated fatty acids (PUFAs) supplemented diets on the proteasome system, in rat skeletal muscles. Thirty rats were randomly assigned to three groups. The control group received only a standard diet. The monounsaturated fatty acid (MUFA) enriched diet group was fed with 3% sunflower oil in addition to standard food, and the polyunsaturated fatty acid supplemented diet group received 9% Maxepa) in addition to the standard diet. We analyzed muscle proteasome activities and content. Monounsaturated or PUFAs supplemented diets given for 8 weeks induced a significant increase in proteasome activities. With the polyunsaturated fatty acid enriched diet, the chymotrypsin-like and peptidylglutamylpeptide hydrolase activities increased by 45% in soleus and extensor digitorum longus (EDL), and by 90% in the gastrocnemius medialis (GM) muscle. Trypsin-like activity of the proteasome increased by 250% in soleus, EDL and GM. This increase in proteasome activities was associated with a concomitant enhancement in the muscle content of proteasome. Proteasome activities and level were less stimulated with a monounsaturated fatty acid supplemented diet. This study provides evidence that a monounsaturated or polyunsaturated fatty acid supplemented diet may regulate muscle proteasomes. Unsaturated fatty acids are particularly prone to free radical attack. Thus, we suggest that alterations in muscle proteasome may result from monounsaturated and polyunsaturated fatty acid-induced peroxidation, in order to eliminate damaged proteins.     

Differential expression of ubiquitin and proteasome-dependent pathway components in rat tissues

The ATP-ubiquitin-dependent pathway in eukaryotes is a complex system, which plays an essential role in selective protein degradation. The functional diversity of this system must be matched to the specific protein metabolism related to the physiology of each cell types. The aim of our work was to study the expression of different components of the proteasome-dependent pathway in various rat tissues. Therefore we quantified the 20S proteasome and the 19S and 11S regulators by Western blot, and measured the expression of the mRNAs of certain subunits, which are markers of these components. We compared the peptidase activities of the purified 20S proteasomes, and also mapped its components by 2D electrophoresis. Our results show that the components of the ATP-ubiquitin-dependent pathway vary considerably both in abundance and activity from one tissue to another. This diversity allows the cells to respond appropriately to tissue-specific protein metabolism in the rat.     

Inactivation of the proteasome by 4-hydroxy-2-nonenal is site specific and dependant on 20S proteasome subtypes

The proteasome represents a major intracellular proteolytic system responsible for the degradation of oxidized and ubiquitinated proteins in both the nucleus and cytoplasm. We have previously reported that proteasome undergoes modification by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) and exhibits declines in peptidase activities during cardiac ischemia/reperfusion. This study was undertaken to characterize the effects of HNE on the structure and function of the 20S proteasome. To assess potential tissue-specific differences in the response to HNE, we utilized purified 20S proteasome from heart and liver, tissues that express different proteasome subtypes. Following incubation of heart and liver 20S proteasome with HNE, changes in the 2D gel electrophoresis patterns and peptidase activities of the proteasome were evaluated. Proteasome subunits were identified by mass spectrometry prior to and following treatment with HNE. Our results demonstrate that specific subunits of the 20S proteasome are targeted for modification by HNE and that modified proteasome exhibits selective alterations in peptidase activities. The results provide evidence for a likely mechanism of proteasome inactivation in response to oxidative stress particularly during cardiac ischemia/reperfusion.