Friedrich Kopp

The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.

Electron microscopy and image analysis of the multicatalytic proteinase

On electron micrographs, negatively stained multicatalytic proteinase molecules are viewed end‐on (ring shaped) or side‐on (rectangular shaped). For aurothioglucose, ammonium molybdate‐ and phosphotungstate‐stained molecules, the dimensions measured are consistent. In contrast, uranyl acetate‐staining reveals ring‐shaped particles which vary in diameter between 12 and 16 nm. This is due to a partial collapse and substantial flattening of the structure. Digital image analysis of side‐on views of the particles reveals a tripartite, reel‐shaped structure. Within the ring‐like, end‐on projections of ammonium molybdate‐stained molecules six local centres of mass can be discerned; their position appears to depart, however, from a true six‐fold symmetry.

Drosophila small cytoplasmic 19S ribonucleoprotein is homologous to the rat multicatalytic proteinase

All eukaryotic cells so far analysed contain 19S particles which share a cylinder-like shape and are composed of a set of proteins of relative molecular mass ranging typically from 19,000 to 36,000 (refs 1–10). Proposed functions have included synthetase activity11, transfer RNA processing12 or messenger RNA repression6, but their biological importance remains obscure. A multicatalytic proteinase (MCP) of similar size and shape has been isolated from mammalian tissues13–24. The apparent similarities of these high molecular weight complexes suggest a biochemical and functional homology between the small cytoplasmic 19S particle from Drosophila melanogaster (19S-scRNP) (ref. 7) and rat MCP (ref. 14). By means of electron microscopy, immunological techniques, RNA identification and proteinase activity assays, we were able to show that the two structurally similar complexes are immunologically related ribonucleoproteins (RNPs) with similar proteolytic activity.

Localization of subunits in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy

The subunit topography of the Thermoplasma acidophilum proteasome was determined by iminunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (α,β). Anti‐α‐subunit IgG was found to bind to the outer disks of the cylinder‐ or barrel‐shaped molecule, while the binding sites of the anti‐β‐subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.

Size and shape of the multicatalytic proteinase from rat skeletal muscle

The multicatalytic proteinase from rat skeletal muscle, a non-lysosomal high molecular weight enzyme active at neutral to alkaline pH, has been examined in the electron microscope as well as by dynamic laser light scattering. Both methods reveal monodisperse particles. Electron micrographs show a cylinder-shaped complex with a diameter of 11 nm and a length of 16 nm in negatively stained, and a diameter of 9.6 nm and a length of 14.3 nm in freeze-dried, heavy metal replicated specimens. The molecule is composed of four rings or disks.

The human proteasome subunit HsN3 is located inthe inner rings of the complex dimer

Subunit HsN3 of the human proteasome is a beta-type subunit homologous to PRE4 from yeast, X1 beta from Xenopus and RN3 from the rat. Using electron microscopy, the binding sites of a monoclonal antibody with specificity for subunit HsN3 have been located in the two juxtaposed inner rings of the human proteasome. Subunit HsN3 was present in two copies, one in each ring, in accordance with our concept of two identical halves making up the complete human proteasome. The subunit is involved in the trypsin-like as well as the peptidylglutamyl-peptide cleavage activities.

Electron microscopy and image analysis reveal common principles of organization in two large protein complexes: groEL-type proteins and proteasomes

In an attempt to settle the question of whether the multicatalytic proteinase or proteasome exist in all three kingdoms of life--eukaryotes, archaebacteria, and eubacteria--we have undertaken a search for them in the eubacterium Comamonas acidovorans. We have, in fact, isolated and purified a cylinder-shaped particle. However, according to various structural and biochemical criteria this turned out to be more reminiscent of the groEL protein from Escherichia coli and its homologs than to proteasomes of eukaryotic or archaebacterial origin. N-terminal sequencing provided definite proof for its belonging to this family of molecular chaperonins. Image analysis of electron micrographs revealed that the C. acidovorans groEL-like protein and proteasomes in spite of their significantly different dimensions have certain principles of organization in common.

In vitro activation of the 20S proteasome.

The effect of chemical compounds like sodium dodecyl sulfate (SDS), fatty acid esters of glycerol, carnitine and coenzyme A, phospholipids, histones, polylysines as well as homobifunctional chemical cross-linkers on the various proteolytic activities of mammalian proteasomes have been tested. Most of the reagents enhance these activities, and some, e.g. fatty acid CoA esters, histones and the chemical cross-linkers, exert dual effects, i.e. activation and inhibition at the same time, depending on the activity measured. With optimally activating concentrations of SDS, no structural changes in proteasomes can be detected by electron microscopy. Formation of micelles at supra-optimal detergent concentrations may be a reason for irreversible denaturation of the proteasome.

Effect of the multicatalytic proteinase (prosome) on translational activity in rabbit reticulocyte lysates

In a message‐dependent reticulocyte lysate translation system, incorporation of [3H]leucine into acid‐insoluble protein is increased following selective removal of the multicatalytic proteinase (MCP) with a monospecific antibody. Re‐addition of active proteinase to previously depleted lysates reverses this effect in that the same low levels of translational product are measured as in untreated lysates. Addition of histone‐stimulated MCP further depresses the level of protein product. Conversely, lysates supplemented with inactivated MCP retain the higher level of translational activity which is measured after precipitation of the enzyme with antibody. In these lysates, the effect of the antibody on translational activity is inversely correlated with that on hydrolytic activity towards [14C]methylcasein or N‐succinyl‐Leu‐Leu‐Val‐Tyr‐4‐methyl‐7‐coumarylamide, two substrates of the MCP. These results showing that the MCP is capable of modulating translational activity in vitro, suggest an important role of this molecule in the in vivo translational process.

Non - Lysosomal, High - Molecular - Mass Cysteine Proteinases from Rat Skeletal Muscle

It is well established that in protein metabolism protein synthesis as well as protein degradation are equally important for maintaining cellular viability. Thus, a basal level of intracellular proteolysis is measurable in living cell 1 , which comprises post-translational processing of newly synthesized proteins 2, degradation of missense proteins 3 as well as breakdown of inactivated proteins having lost their functions 4,5. In addition to the ’basal degradation ‘ an ’accelerated proteolysis‘ takes place in the cells living under conditions of nutrition deprivation as well as hormone or amino acid deficiency 6,7. For catalysis of these various intracellular proteolytic events multiple proteolytic pathways exist in a mammalian cell 8-11. One of the major sites of intracellular proteolysis is the lysosomal compartment. However, since the lysosomal cathepsins have no free access to the protein substrates, initial rate-limiting steps of intracellular proteolysis probably proceed extra-lysosomally. The aim of our investigations is to identify, isolate and characterize the enzymes catalyzing steps of these extra-lysosomal pathways.