Purification and characterization of the 20S proteasome from ostrich skeletal muscle

The proteasome is a high molecular weight, multisubunit and multicatalytic enzyme. Here we report the purification and characterization of ostrich skeletal muscle 20S proteasome. It was purified to homogeneity with Mr 700,000, pI 6.67 and a 'ladder' of 22.2-33.5 kDa bands on SDS-PAGE. The amino acid composition and amino-terminal sequences showed large identities to those of other species. For the three major activities, pH and temperature optima ranged between 8.0-11.0 and 40-70 degrees C, and stabilities between 5-12 and up to 40-60 degrees C. Substrate specificity and inhibitory effects were also studied. Many similarities to other sources were shown, with a few significant differences.     

Developmental regulation of proteolytic activities and subunit pattern of 20 S proteasome in chick embryonic muscle

The proteolytic activities of the 20 S proteasome were found to change in their levels during the development of chick embryonic muscle. The peptide-cleaving activities against N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and N-benzyloxycarbonyl-Ala-Arg-Arg-4-methoxy-beta-naphthylamide gradually decreased with the time of development. On the other hand, the casein-degrading activity in the presence of poly-L-lysine markedly increased from embryonic day 11 and reached a maximal level by day 17. These changes appeared to be tissue-specific because little or no change in any of the proteolytic activities was observed with developing embryonic brain, while dramatic alterations occurred in the extents of the peptide hydrolyses in liver. Furthermore, a number, but not all, of the proteasome subunits in embryonic muscle were changed in their amounts during the development. These results suggest that the alterations in the proteasome activities and subunit pattern are developmentally regulated and may be correlated.     

Structure and structure formation of the 20S proteasome

Eukaryotic 20S proteasomes are complex oligomeric proteins. The maturation process of the 14 different - and -subunits has to occur in a highly coordinate manner. In addition -subunits are synthesized as proproteins and correct processing has to be guaranteed during complex maturation. The structure formation can be subdivided in different phases. The knowledge of the individual phases is summarized in this publication. As a first step the newly synthesized monomers have to adopt the correct tertiary structure, a process that might be supported in the case of the -subunits by the intramolecular chaperone activity postulated for the prosequences. Subsequently the -subunits form ring-like structures thereby providing docking sites for the different -subunits. The result most likely is a double ring structure (13S precursor) representing half-proteasomes, which contain immature proproteins. Two 13S precursors associate to form the proteolytically inactive 16S assembly intermediate which still contains unprocessed -monomers. In addition the chaperone Hsc73 is present within these particles suggesting an essential role during the structure formation process. The processing of monomers with an N-terminal threonine occurs within the 16S particles and is achieved autocatalytically by two subsequent processing events finally leading to the mature, active 20S proteasome.     

The structure of the mammalian 20S proteasome at 2.75 A resolution

The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.     

Dimeric structure of rat skeletal muscle AMP-deaminase subunit

AMP-deaminase from rat skeletal muscle was purified by affinity chromatography on phosphocellulose and gel-filtration on Sephadex G-200. It was established that disulfide bridges and hydrogen bonds were not essential for stability of enzyme oligomeric structure. The dimeric structure of enzyme subunit with Mr 76 kDa (S1) was detected by means of PAGE in the presence of SDS: besides the S1 there were also exhibited two additional bands with Mr 42 (S2) and 33 (S3) kDa. Repeated SDS-PAGE of S1 has revealed the same three protein bands. These results indicate the possibility of dissociation of S1-subunit into two subunits with close Mr values.     

Novel propeptide function in 20 S proteasome assembly influences beta subunit composition

The assembly of eukaryotic 20 S proteasomes involves the formation of half-proteasomes where precursor beta-type subunits gather in position on an alpha-subunit ring, followed by the association of two half-proteasomes and beta-subunit processing. In vertebrates three additional beta-subunits (beta1i/LMP2, beta2i/MECL1, and beta5i/LMP7) can be synthesized and substituted for constitutive homologues (beta1/delta, beta2/Z, and beta5/X) to yield immunoproteasomes, which are important for generating certain antigenic peptides. We have shown previously that when all six beta-subunits are present, cooperative assembly mechanisms limit the diversity of proteasome populations. Specifically, LMP7 is incorporated preferentially over X into preproteasomes containing LMP2 and MECL1. We show here that the LMP7 propeptide is responsible for this preferential incorporation, and it also enables LMP7 to incorporate into proteasomes containing delta and Z. In contrast, the X propeptide restricts incorporation to proteasomes with delta and Z. Furthermore, we demonstrate that the LMP7 propeptide can function in trans when expressed on LMP2, and that its NH(2)-terminal and mid-regions are particularly critical for function. In addition to identifying a novel propeptide function, our results raise the possibility that one consequence of LMP7 incorporation into both immunoproteasomes and delta/Z proteasomes may be to increase the diversity of antigenic peptides that can be generated.     

Novel internally quenched substrate of the trypsin-like subunit of 20S eukaryotic proteasome

This article describes the synthesis, using combinatorial chemistry, of internally quenched substrates of the trypsin-like subunit of human 20S proteasome. Such substrates were optimized in both the nonprime and prime regions of the peptide chain. Two were selected as the most susceptible for proteasomal proteolysis with excellent kinetic parameters: (i) ABZ-Val-Val-Ser-Arg-Ser-Leu-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 934,000 M⁻¹ s⁻¹) and (ii) ABZ-Val-Val-Ser-GNF-Ala-Met-Gly-Tyr(3-NO₂)-NH₂ (k_cat/K_M = 1,980,000 M⁻¹ s⁻¹). Both compounds were efficiently hydrolyzed by the 20S proteasome at picomolar concentrations, demonstrating significant selectivity over other proteasome entities.     

Differential effects of detergents, fatty acids, cations and heating on ostrich skeletal muscle 20S proteasome

The 20S proteasome, the catalytic core of the 26S proteasome, has previously been isolated, purified and partially characterised from ostrich skeletal muscle (Thomas, A.R., Oosthuizen, V., Naude, R.J., Muramoto, K. 2002. Biol. Chem. 383, 1267-1270). Due to the apparent latency of the 20S proteasome purified from various sources, this study focuses on further characterising the ostrich enzyme in terms of the effects of selected detergents, fatty acids and cations, as well as heating at 60 degrees C, on four of its activities. Results showed that ostrich skeletal muscle 20S proteasome was affected in a non-concentration-dependent manner by the selected detergents and fatty acids. Monounsaturated fatty acids, unlike unsaturated fatty acids, showed no major effects on the activities of the ostrich enzyme. The enzyme did not show sensitivity towards monovalent cations and the only divalent cations that showed a relevant effect were Ca2+ and Mg2+. Heating at 60 degrees C for 1-2 min had a substantial activating effect only on the peptidylglutamylpeptide-hydrolase (PGPH) and caseinolytic activities. In conclusion, many of the effects by the abovementioned reagents and conditions were noticeably different to those shown on different sources of the enzyme, further demonstrating the unique kinetic characteristics of the ostrich skeletal muscle 20S proteasome.     

The 60S ribosomal subunit is altered in the skeletal muscle of dystrophic hamsters

Polysomes from the skeletal muscle of normal and dystrophic hamsters were dissociated into ribosomal subunits by treatment with puromycin and the subunits from both strains were reassociated in all possible combinations. When their protein synthesis activity was assayed in a poly(U)-directed cell-free system at a low magnesium concentration, the reassociated ribosomes from dystrophic hamsters were less active than the ribosomes from control animals. The ribosomal defect is a property of the 60S subunit and is due to a ribosomal component rather than to abnormal binding of a non-ribosomal protein.     

Alteration of 20S proteasome-subtypes and proteasome activator PA28 in skeletal muscle of rat after induction of diabetes mellitus

Insulin-dependent diabetes mellitus is known to go along with enhanced muscle protein breakdown. Since evidence has been presented that the ubiquitin-proteasome system is significantly involved in muscle wasting under this condition, we have investigated, whether this biological role goes along with alterations of the proteasome system in skeletal muscle of streptozotocin-diabetic rats. Previously, we have found a drop of overall proteasome activity in muscle extracts of rats after induction of diabetes but no change in total amount of 20S proteasome was detected. In the present investigation under the same diabetic conditions we have measured a significant decrease in the amount of proteasome activator PA28, a finding that explains the loss of total proteasome activity. Since increased mRNA levels of proteasome subunits have been measured in muscle tissue of rats after induction of diabetes, we have isolated and purified 20S proteasomes from muscle tissue of control and 6 days diabetic rats. The specific chymotrypsin-like, trypsin-like, and peptidylglutamylpeptide-hydrolysing activities of proteasomes from diabetic and control rats were found to be not significantly different. Therefore, we have fractionated 20S proteasomes into their subtypes and detected that induction of diabetes mellitus effects a redistribution of subtypes of all three proteasome populations but only the increase in subtype V (immuno-subtype) was statistically significant. This altered subtype pattern obviously meets the requirements to the system under wasting conditions. Since this process goes along with de novo biogenesis of 20S proteasomes, it most likely explains the phenomenon of elevated mRNA concentrations of proteasome subunits after induction of diabetes mellitus.     

Biogenesis, structure and function of the yeast 20S proteasome.

Intracellular degradation of many eukaryotic proteins requires their covalent ligation to ubiquitin. We previously identified a ubiquitin-dependent degradation pathway in the yeast Saccharomyces cerevisiae, the DOA pathway. Independent work has suggested that a major mechanism of cellular proteolysis involves a large multisubunit protease(s) called the 20S proteasome. We demonstrate here that Doa3 and Doa5, two essential components of the DOA pathway, are subunits of the proteasome. Biochemical analyses of purified mutant proteasomes suggest functions for several conserved proteasome subunit residues. All detectable proteasome particles purified from doa3 or doa5 cells have altered physical properties; however, the mutant particles contain the same 14 different subunits as the wild-type enzyme, indicating that most or all yeast 20S proteasomes comprise a uniform population of hetero-oligomeric complexes rather than a mixture of particles of variable subunit composition. Unexpectedly, we found that the yeast Doa3 and Pre3 subunits are synthesized as precursors which are processed in a manner apparently identical to that of related mammalian proteasome subunits implicated in antigen presentation, suggesting that biogenesis of the proteasome particle is highly conserved between yeast and mammals.     

Subtypes of 20S proteasomes from skeletal muscle

20S proteasomes from tissues and cells are a mixture of several subtypes. From rat skeletal muscle we have tentatively separated six different subtypes of 20S proteasomes purified from rat skeletal muscle by high-resolution anion exchange chromatography. Immunoblot analysis using antibodies to the beta-subunits LMP2, LMP7 and their constitutive counterparts delta and MB1 revealed that two of the three major subtypes (subtypes I and II) are constitutive proteasomes, whereas two of the three minor subtypes belong to the subpopulation of immuno-proteasomes. Subtype III and IV are intermediate-type proteasomes. Enzymological characterisation of the six subtypes revealed clearly different V(max) values for hydrolysis of fluorogenic peptide substrates as well as significantly different activities measured with a 25-mer polypeptide of the murine cytomegalovirus IE pp89 protein as substrate. Our data show that the properties of 20S proteasomes isolated from a given tissue or cells are always the average of the properties of the whole set of proteasome subtypes.     

Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome

The dipeptide boronic acid bortezomib, also termed VELCADE, is a proteasome inhibitor now in use for the treatment of multiple myeloma, and its use for the treatment of other malignancies is being explored. We determined the crystal structure of the yeast 20S proteasome in complex with bortezomib to establish the specificity and binding mode of bortezomib to the proteasome's different catalytically active sites. This structure should enable the rational design of new boronic acid derivatives with improved affinities and specificities for individual active subunits.     

Assembly of the 20S proteasome

The proteasome is a cellular protease responsible for the selective degradation of the majority of the intracellular proteome. It recognizes, unfolds, and cleaves proteins that are destined for removal, usually by prior attachment to polymers of ubiquitin. This macromolecular machine is composed of two subcomplexes, the 19S regulatory particle (RP) and the 20S core particle (CP), which together contain at least 33 different and precisely positioned subunits. How these subunits assemble into functional complexes is an area of active exploration. Here we describe the current status of studies on the assembly of the 20S proteasome (CP). The 28-subunit CP is found in all three domains of life and its cylindrical stack of four heptameric rings is well conserved. Though several CP subunits possess self-assembly properties, a consistent theme in recent years has been the need for dedicated assembly chaperones that promote on-pathway assembly. To date, a minimum of three accessory factors have been implicated in aiding the construction of the 20S proteasome. These chaperones interact with different assembling proteasomal precursors and usher subunits into specific slots in the growing structure. This review will focus largely on chaperone-dependent CP assembly and its regulation.     

Identification of the goldfish 20S proteasome beta6 subunit bound to nuclear matrix

Proteasomes are large, multisubunit particles that act as the proteolytic machinery for most of the regulated intracellular protein breakdown in eukaryotic cells. Proteasomes are present in both the nucleus and cytoplasm. When we analyzed the molecular composition of protein constituents of the nuclear matrix preparation of goldfish oocytes by two-dimensional polyacrylamide gel electrophoresis followed by sequence analysis, we found a 26 kDa spot identical in amino acid sequence to the beta6 subunits of the 20S proteasome. No spot of other subunits of 20S proteasome was detected. Here we describe the cloning, sequencing and expression analysis of Carassius auratus, beta6_ca, which encodes one of the proteasome beta subunits from goldfish ovary. From the screening of an ovarian cDNA library, two types of cDNA were obtained, one 941 bp and the other 884 bp long. The deduced amino acid sequences comprise 239 and 238 residues, respectively. These deduced amino acid sequences are highly homologous to those of beta6 subunits of other vertebrates. Immunoblot analysis of nuclear matrix using anti-proteasome antibodies showed only a spot of beta6_ca. These results suggest that the beta6 subunit of the goldfish 20S proteasome, beta6_ca, is responsible for anchoring proteasomes in the nucleus.     

The pertussis toxin S1 subunit is a thermally unstable protein susceptible to degradation by the 20S proteasome

Pertussis toxin (PT) is an AB-type protein toxin that consists of a catalytic A subunit (PT S1) and an oligomeric, cell-binding B subunit. It belongs to a subset of AB toxins that move from the cell surface to the endoplasmic reticulum (ER) before the A chain passes into the cytosol. Toxin translocation is thought to involve A chain unfolding in the ER and the quality control mechanism of ER-associated degradation (ERAD). The absence of lysine residues in PT S1 may allow the translocated toxin to avoid ubiquitin-dependent degradation by the 26S proteasome, which is the usual fate of exported ERAD substrates. As the conformation of PT S1 appears to play an important role in toxin translocation, we used biophysical and biochemical methods to examine the structural properties of PT S1. Our in vitro studies found that the isolated PT S1 subunit is a thermally unstable protein that can be degraded in a ubiquitin-independent fashion by the core 20S proteasome. The thermal denaturation of PT S1 was inhibited by its interaction with NAD, a donor molecule used by PT S1 for the ADP ribosylation of target G proteins. These observations support a model of intoxication in which toxin translocation, degradation, and activity are all influenced by the heat-labile nature of the isolated toxin A chain.     

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.