Burkhardt Dahlmann

The base of the proteasome regulatory particle exhibits chaperone-like activity

Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme. Protein folding in vivo is mediated by molecular chaperones. Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase. Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase. We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel. Denatured but not native citrate synthase is bound by the base complex. Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome. These data suggest a model in which ubiquitin–protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

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.

Role of proteasomes in disease.

A functional ubiquitin proteasome system is essential for all eukaryotic cells and therefore any alteration to its components has potential pathological consequences. Though the exact underlying mechanism is unclear, an age-related decrease in proteasome activity weakens cellular capacity to remove oxidatively modified proteins and favours the development of neurodegenerative and cardiac diseases. Up-regulation of proteasome activity is characteristic of muscle wasting conditions including sepsis, cachexia and uraemia, but may not be rate limiting. Meanwhile, enhanced presence of immunoproteasomes in aging brain and muscle tissue could reflect a persistent inflammatory defence and anti-stress mechanism, whereas in cancer cells, their down-regulation reflects a means by which to escape immune surveillance. Hence, induction of apoptosis by synthetic proteasome inhibitors is a potential treatment strategy for cancer, whereas for other diseases such as neurodegeneration, the use of proteasome-activating or -modulating compounds could be more effective.Publication history: Republished from Current BioDatas Targeted Proteins database (TPdb; http://www.targetedproteinsdb.com).

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.

Extracellular, circulating proteasomes and ubiquitin — Incidence and relevance

The ubiquitin-proteasome system is the major pathway for intracellular protein degradation and is also deeply involved in the regulation of most basic cellular processes. Its proteolytic core, the 20S proteasome, has found to be attached also to the cell plasma membrane and certain observations are interpreted as to suggest that they may be released into the extracellular medium, e.g. in the alveolar lining fluid, epididymal fluid and possibly during the acrosome reaction. Proteasomes have also been detected in normal human blood plasma and designated circulating proteasomes; these have a comparatively low specific activity, a distinct pattern of subtypes and their exact origin is still enigmatic. In patients suffering from autoimmune diseases, malignant myeloproliferative syndromes, multiple myeloma, acute and chronic lymphatic leukaemia, solid tumour, sepsis or trauma, respectively, the concentration of circulating proteasomes has been found to be elevated, to correlate with the disease state and has even prognostic significance. Similarly, ubiquitin has been discovered as a normal component of human blood and seminal plasma and in ovarian follicular fluid. Increased concentrations were measured in diverse pathological situations, not only in blood plasma but also in cerebrospinal fluid, where it may have neuroprotective effects. As defective spermatozoa are covered with ubiquitin in the epididymal fluid, extracellular ubiquitination is proposed to be a mechanism for quality control in spermatogenesis. Growing evidence exists also for a participation of extracellular proteasomes and ubiquitin in the fertilization process.

The three-dimensional structure of proteasomes from Thermoplasma acidophilum as determined by electron microscopy using random conical tilting

The three‐dimensional structure of proteasomes from the archaebacterium Thermoplasma acidophilum has been determined to a resolution of approximately 2 nm from electron micrographs of negatively stained preparations using the method of random conical tilting. The particles turn out to be essentially cylinder‐shaped barrels, 15 nm long and 11 nm wide, enclosing a tripartite inner compartiment. An account is given of some of the present limitations which prevent to attain a higher resolution and possible ways to overcome these limitations are indicated.

Cloning and sequencing of the gene encoding the large (α-) subunit of the proteasome from Thermoplasma acidophilum

The gene encoding the α‐subunit of the proteasome from Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid and reduces a calculated molecular weight of 25870. Sequence similarity of the α‐subunit with the Saccharomyces cerevislae wild‐type suppressor gene sell* encoded polypeptide, which is probably identical with the subunit YC7‐α of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.

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.