Mariele Briand

PROTEOLYTIC ACTIVITY OF PROTEASOME ON MYOFIBRILLAR STRUCTURES

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than α-actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.

Brain proteasomal function in sporadic Parkinson's disease and related disorders

Because genetic defects relating to the ubiquitin–proteasome system were reported in familial parkinsonism, we evaluated proteasomal function in autopsied brains with sporadic Parkinsons disease. We found that proteasome peptidase activities in a fraction specific to the proteasome were preserved in five brain areas (including the striatum) of Parkinsons disease where neuronal loss is not observed. Striatal protein levels of two proteasome subunits were normal in Parkinsons disease but reduced mildly in disease controls (multiple system atrophy). Our brain data suggest that a systemic, global disturbance in the catalytic activity and degradation ability of the proteasome itself is unlikely to explain the cause of Parkinsons disease.

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.

The effect of proteasome on myofibrillar structures in bovine skeletal muscle

When bovine myofibrils are incubated with the 20S proteasome their structure is rapidly damaged with loss of material, particularly from the Z discs and I bands. After 24 hr of incubation the myofibrils rupture and debris appears. Certain myofibrillar proteins, including nebulin, myosin, actin and tropomyosin, are hydrolysed during the incubation; others are solubilised (α-actinin). The 20S proteasome completely and rapidly hydrolyses purified myofibrillar proteins in an energy-independent manner. This shows that the 20S proteasome probably plays a role in the postmortem transformation of muscle and more generally in the hydrolysis of cellular proteins.(1).

Role of Calpains in Postmortem Proteolysis in Chicken Muscle

Tenderness is governed by postmortem biochemical processes, particularly proteolysis. In mammals, the calpain system is generally accepted as the main system involved in postmortem proteolysis. In poultry, the 2 calpains (mu and mu/m--a form only found in bird tissue) have greater calcium sensitivity. In this study, we quantified by zymography the changes in postmortem calpain system activity. The mu/m-calpain activity remained steady, whereas the mu-calpain activity had disappeared by 6 h after postmortem, showing an activation by calcium. Changes in the electrophoretic pattern of sarcoplasmic and myofibrillar proteins are observed in the first postmortem hours concomitantly to the decrease in mu-calpain activity. The 30-kDa protein, considered as a good marker of postmortem aging in cattle, appeared from 6 h and then steadily increased. In chicken muscle, the rapid maximum tenderness reached could be explained by a greater activation of the calpain system.

Linkage between the proteasome pathway and neurodegenerative diseases and aging

During aging, the production of free radicals increases. This can result in damage to protein, the accumulation of which is characteristic of the aging process. This questions the efficacy of proteolytic systems. Among these systems, the proteasome and the adenosine triphosphate-ubiquitin-dependent pathway have been shown to play an important role in the elimination of abnormal proteins. There are two major steps in the ubiquitin-proteasome pathway: the conjugation of a polyubiquitin degradation signal to the substrate and the subsequent degradation of the tagged protein by the 26S proteasome. The 26S proteasome is build-up from the 20S proteasome, which is a cylinder-shaped multimeric complex, and two additional 19S complexes. The 20S proteasome can also bind to 11S regulator and is then implicated in antigen presentation. These regulators confer a high adaptability on proteasome.With advancing age, predisposition to neurodegenerative diseases increases. These diseases are also characterized by protein aggregation. Several findings such as the presence of ubiquinated proteins, usually broken down by proteasomes, and genetic anomalies involving the ubiquitinproteasome system (parkin, UCH-L1) suggest a link between the ubiquitin-proteasome pathway and the genesis of these diseases.

Metabolic and contractile differentiation of rabbit muscles during growth

1. A study was carried out of post-natal evolution of the oxidative, glycolytic and contractile capacities in various types of rabbit muscle. 2. At birth, muscles are non-differentiated and present very limited metabolic and contractile activity, metabolism is mainly oxidative in all muscles. 3. Although muscular discrimination is manifest from the sixth week after birth, the glycolytic metabolism reaches its maximum capacity only after six to eight weeks. 4. Subsequently, oxidative metabolic capacity steadily decreases until adulthood.

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 Parkinsons and Alzheimers 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.

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.

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.