Structure and assembly of the 20S proteasome.

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain alpha-type subunits, the inner ones comprise beta-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the alpha-type and beta-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the beta-type subunits are processed. The prosequence of the beta-type subunits is essential for the assembly proves and prevents protease activity of immature proteasomes.

The human proteasomal subunit HsC8 induces ring formation of other alpha-type subunits.

The eukaryotic 20 S proteasome is a barrel-shaped protease complex, made up of four seven-membered rings. The outer and inner rings contain seven different alpha and beta-type subunits, respectively, each subunit located at a defined position. Recently, we have reported that the recombinant human alpha-type subunit C8 (HsC8) assembles into a heptameric ring-like structure by itself. In the present study we show that the two naturally neighboring alpha-type subunits of HsC8, HsPROS30 and HsPROS27, do not form ring-like complexes by themselves, but only dimers. This indicates that the propensity to form homo-oligomeric rings is not a general feature among human alpha-type subunits. However, coexpression of HsC8 and either of these neighbor alpha-type subunits results in the formation of hetero-oligomeric ring complexes, resembling the HsC8 ring-like structure. The ratio between the two types of subunits in the mixed complexes is surprisingly heterogeneous, varying from very high to very low HsC8 content. The three tested alpha-type subunits thus apparently lack binding sites that selectively interact with a specific neighboring subunit. This suggests that the correct positioning of the different alpha-type subunits in the eukaryotic 20 S proteasome is not dictated by the alpha-type subunits themselves, but rather by the interaction with specific beta-type subunits.

Detailed analysis of cell cycle kinetics upon proteasome inhibition.

We have studied specific effects of proteasome inhibition on cell cycle progression. To this end, the protease inhibitors MG115, calpain inhibitor I, and calpain inhibitor II, which display differential inhibitory effects on proteasomes, were used. Cell kinetic studies using bromodeoxyuridine pulse labeling revealed a complete block of G1/S and metaphase transitions and a delayed progression through S phase in cell cultures treated with 54 microM of MG115. Calpain inhibitor I in similar concentrations displayed a fivefold lower effect on cell cycle kinetics. Calpain inhibitor II and MG2M, which is a structural analogue of MG115, had no effect on the cell cycle. The inhibitory effect of MG115 treatment was reversible, because the cell cycle was immediately resumed when the MG115-containing culture medium was replaced by fresh culture medium. Because ubiquitinated proteins accumulated after MG115 treatment, it was confirmed that ubiquitin-dependent protein degradation, and thus proteasomal activity were blocked. By comparison of biochemical and in vitro proteasome inhibition experiments, it was hypothesized that chymotrypsin-like activity of proteasomes may play an important role in cell cycle kinetics.

The human alpha-type proteasomal subunit HsC8 forms a double ringlike structure, but does not assemble into proteasome-like particles with the beta-type subunits HsDelta or HsBPROS26.

The eukaryotic proteasome is a barrel-shaped protease complex made up of four seven-membered rings of which the outer and inner rings may contain up to seven different alpha- and beta-type subunits, respectively. The assembly of the eukaryotic proteasome is not well understood. We cloned the cDNA for HsC8, which is one of the seven known human alpha-type subunits, and produced the protein in Escherichia coli. Recombinant HsC8 protein forms a complex of about 540 kDa consisting of double ringlike structures, each ring containing seven subunits. Such a structure has not earlier been reported for any eukaryotic proteasome subunit, but is similar to the complex formed by the recombinant alpha-subunit of the archaebacterium Thermoplasma acidophilum (Zwickl, P., Kleinz, J., and Baumeister, W. (1994) Nat. Struct. Biol. 1, 765-770). The ability of HsC8 to form alpha-rings suggests that these complexes may play an important role in the initiation of proteasome assembly in eukaryotes. To test this, we used two human beta-type subunits, HsBPROS26 and HsDelta. Both these beta-type subunits, either in the proprotein or in the mature form, exist in monomers up to tetramers. In contrast to the alpha- and beta-subunit of T. acidophilum, coexpression of the human beta-type subunits with HsC8 does not result in the formation of proteasome-like particles, which would be in agreement with the notion that proteasome assembly in eukaryotes is much more complex than in archaebacteria.

Reduced chaperone-like activity of alpha A(ins)-crystallin, an alternative splicing product containing a large insert peptide.

alpha-Crystallin is a multimeric protein complex which is constitutively expressed at high levels in the vertebrate eye lens, where it serves a structural role, and at low levels in several non-lenticular tissues. Like other members of the small heat shock protein family, alpha-crystallin has a chaperone-like activity in suppressing nonspecific aggregation of denaturing proteins in vitro. Apart from the major alpha A- and alpha B-subunits, alpha-crystallin of rodents contains an additional minor subunit resulting from alternative splicing, alpha A(ins)-crystallin. This polypeptide is identical to normal alpha A-crystallin except for an insert peptide of 23 residues. To explore the structural and functional consequences of this insertion, we have expressed rat alpha A- and alpha A(ins)-crystallin in Escherichia coli. The multimeric particles formed by alpha A(ins) are larger and more disperse than those of alpha A, but they are native-like and display a similar thermostability and morphology, as revealed by gel permeation chromatography, tryptophan fluorescence measurements, and electron microscopy. However, as compared with alpha A, the alpha A(ins)-particles display a diminished chaperone-like activity in the protection of heat-induced aggregation of beta low-crystallin. Our experiments indicate that alpha A(ins)-multimers have a 3-4-fold reduced substrate binding capacity, which might be correlated to their increased particle size and to a shielding of binding sites by the insert peptides. The structure-function relationship of the natural mutant alpha A(ins)-crystallin may shed light on the mechanism of chaperone-like activity displayed by all small heat shock proteins.

Cloning and expression of a human pro(tea)some beta-subunit cDNA: a homologue of the yeast PRE4-subunit essential for peptidylglutamyl-peptide hydrolase activity.

The cDNA encoding a human prosome beta-subunit (HSBpros26) was isolated from a lymphoma library using the cDNA of the Xenopus homologue as a probe. The cDNA contains an open reading frame encoding a protein of 233 amino acids and a calculated molecular weight of 25,909. Comparison with interspecies homologues of HSBpros26 from Xenopus (XLB), rat (RN3) and yeast (PRE4) reveals a high degree of identity between the beta-subunits except for the N-terminal end, which is probably cleaved post-translationally. The complete coding sequence of HSBpros26 has been expressed in E. coli. The produced protein of about 27 kDa reacts with the prosomal monoclonal antibody MCP205, kindly provided by Dr. K. Hendil. The molecular weight of the native protein is about 28 kDa indicating that the protein is present as monomers. Finally partially purified HSBpros26 preparations do not contain any proteolytical activity.

Biochemical and structural aspects of transiently and stably expressed mutant desmin in vimentin-free and vimentin-containing cells.

Using immunoelectron microscopy it is demonstrated that desmin subunits missing their complete carboxy-terminal domain are incapable of homopolymeric filament formation in vivo. Furthermore it is shown that, in vimentin-containing cells, desmin integrates into preexisting vimentin filaments resulting in desmin/vimentin heteropolymers. Removal of the amino-terminal or both nonhelical end domains of desmin increases Triton X-100 solubility of the mutant desmin subunits. Expression of desmin mutants containing deletions in the C-terminal part of the rod in vimentin-free cells results in an increase of the Triton X-100 solubility too. In contrast, if expressed in vimentin-containing cells, these mutant subunits remain in the Triton X-100 insoluble fraction. Deletion of the nonhelical carboxy-terminal domain only has no effect on solubility. In vimentin-free cells, stably expressed desmin subunits missing their amino-terminal domains display a slightly higher turnover rate compared to wild-type desmin. Transiently expressed desmin subunits missing 18 or more carboxy-terminal residues of the rod domain are rapidly degraded in vimentin-free cells. In vimentin-containing cells, turnover rates were much less pronounced. Finally, by using site-directed mutagenesis, we were able to map specific residues important for de novo filament assembly within the amino-terminal domain and in the conserved part at the C-terminus of the alpha-helical domain.

Assembly of carboxy-terminally deleted desmin in vimentin-free cells.

Using a vimentin-free expression system we were able to demonstrate that the carboxy terminus of desmin is necessary for filament assembly in the living cell. Desmin subunits missing only 4 carboxy-terminal residues of their rod domain are incapable of homopolymeric filament assembly. Moreover, even single amino acid substitutions in the conserved carboxy-terminal part of the rod domain prevent desmin subunits from homopolymeric filament assembly. Desmin subunits missing 18 or more carboxy-terminal residues of their rod domain (including the complete conserved carboxy-terminal region) are unstable in cells devoid of intact type III intermediate filaments (IFs). Interaction with an intact type III IF, however, stabilizes these mutated desmin subunits. Expression of a desmin subunit missing both its non-helical end domains in vimentin-containing cells disrupts the endogenous vimentin network completely.