Yves 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.

Identification and Initial Characterization of a Specific Proteasome (Prosome) Associated RNase Activity

We have identified and characterized a specific nuclease activity to be tightly associated with proteasomes. Using tobacco mosaic virus RNA (TMV-RNA) as substrate to analyze and quantify the cleavage reaction, we supply several lines of evidence that this nuclease activity is an integral part of proteasomes. Thus, RNase activity was coincident with the elution profiles of proteasomes at each stage of purification. Proteasomal nuclease activity was resistant to strong dissociation conditions using 480 mM KCl, 0.5% sodium lauroylsarcosinate, and 6 M urea. This nuclease activity remained associated with an urea-resistant subcomplex of the proteasome comprising a specific set of proteins. Finally the digestion of TMV-RNA led to a well defined pattern of RNA fragments while 5 S ribosomal RNA and globin mRNA were not degraded. These results provide further evidence that proteasomes are able to discriminate between different RNAs, and the possible involvement of proteasomes in translation control is discussed.

Proteasome (prosome) associated endonuclease activity

The 20S proteasome (prosome) is a highly organized multiprotein complex with approximate molecular weight of about 700 kDa. Whilst the role of the proteasome in the processing and turnover of cellular proteins is becoming clearer, its relationship with RNA remains still obscure. Here we focus on the nature and function of proteasome associated endonuclease activity. Thus the involvement of a proteasome α-type subunit in RNA-degradation, the catalytic requirements, the interaction of proteasomes with their RNA-substrate and the identification of a well defined cleavage site in the 3-UTR of short-lived cellular mRNAs will be described in detail. All data indicate that proteasomes associated endonuclease activity could be involved in post-transcriptional gene control at the level of translation. Abbreviations: Hu – human; Mu – murine; β IFN – fibroblast interferon; γ IFN – lymphoblast interferon; c FOS – fos proto-oncogene; G-CSF – granulocyte colony stimulating factor.

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.

Proteasomes (prosomes) inhibit the translation of Tobacco mosaic virus RNA by preventing the formation of initiation complexes

Proteasomes (prosomes) are large multiprotein complexes. They are involved in protein degradation of ubiquitin-conjugated proteins and in the generation of MHC class I peptides. We gave further evidence that they interfere within vitro protein synthesis. Proteasomes inhibit the translation of Tobacco mosaic virus RNA. Analysis of cell-free systems by sucrose gradient centrifugation revealted that they prevent the formation of 80S initiation complexes but not the early phase of initiation.