Subunit topology of two 20S proteasomes from Haloferax volcanii

Haloferax volcanii, a halophilic archaeon, synthesizes three different proteins (alpha1, alpha2, and beta) which are classified in the 20S proteasome superfamily. The alpha1 and beta proteins alone form active 20S proteasomes; the role of alpha2, however, is not clear. To address this, alpha2 was synthesized with an epitope tag and purified by affinity chromatography from recombinant H. volcanii. The alpha2 protein copurified with alpha1 and beta in a complex with an overall structure and peptide-hydrolyzing activity comparable to those of the previously described alpha1-beta proteasome. Supplementing buffers with 10 mM CaCl(2) stabilized the halophilic proteasomes in the absence of salt and enabled them to be separated by native gel electrophoresis. This facilitated the discovery that wild-type H. volcanii synthesizes more than one type of 20S proteasome. Two 20S proteasomes, the alpha1-beta and alpha1-alpha2-beta proteasomes, were identified during stationary phase. Cross-linking of these enzymes, coupled with available structural information, suggested that the alpha1-beta proteasome was a symmetrical cylinder with alpha1 rings on each end. In contrast, the alpha1-alpha2-beta proteasome appeared to be asymmetrical with homo-oligomeric alpha1 and alpha2 rings positioned on separate ends. Inter-alpha-subunit contacts were only detected when the ratio of alpha1 to alpha2 was perturbed in the cell using recombinant technology. These results support a model that the ratio of alpha proteins may modulate the composition and subunit topology of 20S proteasomes in the cell.     

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.     

Development of lipopeptides for inhibiting 20S proteasomes

Proteasomes are responsible for the cytoplasmic turnover of the vast majority of proteins including regulatory proteins. We have synthesized lipopeptides a new class of non-covalent inhibitors of the 20S proteasome and assayed their inhibitory capacities. Their ability to inhibit at micromolar concentrations chymotrypsin-like and post-acid activities depends on peptide length (3 or 6 amino acids), sequence (presence of a positively or negatively charged amino acid), and alkyl chain length (C6-C18). These structural features could be varied to selectively inhibit one or more of the three proteasome activities.     

Multiple chaperone-assisted formation of mammalian 20S proteasomes

Protein degradation is essential for maintenance of cellular homeostasis. The majority of proteins are selectively degraded in eukaryotic cells by the ubiquitin-proteasome system. The 26S proteasome selects target proteins that are covalently modified with polyubiquitin chains. The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. The catalytic activities are carried out by the core 20S proteasome. The eukaryotic 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7. Recent studies have revealed the mechanism responsible for the assembly of such a complex structure. This article recounts the observations that disclosed the biogenesis of 20S proteasomes and discusses the difference in the mechanism of assembly between archael, yeast, and mammalian 20S proteasomes.     

Late events in the assembly of 20S proteasomes

Electron microscopy and STEM mass measurements have been used to characterize late intermediates in the assembly pathway of wildtype and mutant Rhodococcus proteasomes. A proteolytically inactive and processing-incompetent mutant, betaK33A, allowed a short-lived late intermediate of the pathway to be captured, the preholoproteasome. In this fully assembled 20S complex the 14 propeptides with an aggregate mass of 100 kDa fill the whole central cavity and most of the two antechambers. It is further shown that in wildtype Rhodococcus proteasomes the propeptides are degraded in a processive manner undergoing multiple cleavages before the products are discharged and the inner cavities are cleared. It appears that the docking of two half-proteasomes, i.e., preholoproteasome formation, is sufficient to trigger autocleavage of the Gly-1/Thr1 bond necessary for active site formation and the subsequent degradation of the propeptides.     

The complete primary structure of mouse 20S proteasomes

 The proteasome is a large multicatalytic proteinase that plays a role in the generation of peptides for presentation by major histocompatibility complex class I molecules. The 20S proteolytic core of mammalian proteasomes is assembled from a group of 17 protein subunits that generate a distinctive pattern of spots upon two-dimensional gel electrophoresis. The genes for most of these subunits have been cloned from humans and rats. We isolated cDNA clones for the mouse orthologues of ten of the subunits [PSMA1 (C2), PSMA2 (C3), PSMA3 (C8), PSMA4 (C9), PSMA5 (ZETA), PSMA6 (IOTA), PSMA7 (C6-I), PSMB2 (C7-I), PSMB3 (C10-II), and PSMB5 (X)] to complete the cloning of all of the mouse subunits. Using antisera raised against these subunits or their orthologues, we verified the identity of these proteins by two-dimensional NEPHGE-PAGE. Received: 8 March 1999 / Accepted: 8 April 1999     

Beyond cells: The extracellular circulating 20S proteasomes

Accumulating evidence arising from numerous clinical studies indicate that assembled and functional 20S proteasome complexes circulate freely in plasma. Elevated levels of this core proteolytic complex have been found in the plasma of patients suffering from blood, skin and solid cancers, autoimmune disorders, trauma and sepsis. Moreover, in various diseases, there is a positive correlation between circulating 20S proteasome (c20S) levels and treatment efficacy and survival rates, suggesting the involvement of this under-studied c20S complex in pathophysiology. However, many aspects of this system remain enigmatic, as we still do not know the origin, biological role or mechanisms of extracellular transport and regulation of c20S proteasomes. In this review, we provide an overview of the current understanding of the c20S proteasome system and discuss the remaining gaps in knowledge.     

The effect of heat shock on 20S/26S proteasomes

We have studied the consequences of heat shock on 20S/26S proteasome activity and activation, the proteasomal subunit composition, proteasome assembly, subunit mRNA stability as well as on the intracellular distribution of proteasomes. Our data show that heat shock locks 20S proteasomes in their latent inactive state and impairs further activation of the 26S proteasome by ATP. Proteasome mRNA levels are decreased after heat shock and the assembly of the proteasome complex is inhibited. Heat shock also induces a rapid reorganisation of the cellular distribution of the proteasome which appears to be connected with proteasome activity and the change of the cellular architecture after heat shock.     

Identical subunit topographies of human and yeast 20S proteasomes

The arrangement of subunits in human 20S proteasomes was recently determined by us by immunoelectron microscopy and chemical cross-linking. The positions of 4 of the 14 subunits differed from those found in the yeast proteasome by X-ray crystallography. Double labeling of human 20S proteasomes with antibodies to subunits C2 and C5 has now shown that these subunits are nearest neighbors. The result contradicts our published model for the human proteasome but is in accordance with the subunit arrangement in yeast proteasomes, suggesting that yeast and human proteasomes most probably have identical subunit arrangements. Immunoelectron microscopy also showed that the C-terminal extension at the human C2 subunit is flexible but takes up a well-defined position in the proteasome.     

Wheat sprout extract induces changes on 20S proteasomes functionality

Wheat sprouts contain a very high level of organic phosphates and a powerful cocktail of different molecules such as enzymes, reducing glycosides and polyphenols. The antioxidant properties of wheat sprouts have been widely documented and it has been shown that they are able to protect DNA against free-radicals mediated oxidative damage. Furthermore, we have recently reported on the effects of several polyphenols on 20S proteasomes, underlying the dual role of epigallocatechin-3-gallate as an antioxidant and a proteasome effector in cancer cells. The aim of this study was to investigate the effects of wheat sprout extracts on 20S proteasome functionality. Wheat sprout extracts have been analysed and characterized for their polyphenolic content using the Folin-Ciocalteau reagent and RP-HPLC technique. Comparing our data with a polyphenol standard mixture we identified five different polyphenols: gallic acid, epigallocatechin-3-gallate, epigallocatechin, epicatechin and catechin. The treatment of isolated 20S proteasomes with the extract induced a gradual inhibition of all the tested components, ChT-L, T-L, PGPH and BrAAP, in both the complexes. At low extract concentration a slight activation of the enzyme was evident only for the BrAAP component of the constitutive enzyme and the ChT-L activity of the immunoproteasome. beta-casein degradation rate decreased, particularly with the immunoproteasome. Human Colon adenocarcinoma (Caco) cells, stimulated with 12-O-tetradecanoylphorbol-13-acetate, showed activation of the 20S proteasome activities at short incubation times and an increase in intracellular oxidative proteins. Cells treatment with wheat sprout extract led to proteasome inhibition in unstimulated cells and attenuated the effects mediated by TPA. Finally, exposure to the extract affected the expression levels of pro-apoptotic proteins.     

20S proteasomes and protein degradation "by default"

The degradation of the majority of cellular proteins is mediated by the proteasomes. Ubiquitin-dependent proteasomal protein degradation is executed by a number of enzymes that interact to modify the substrates prior to their engagement with the 26S proteasomes. Alternatively, certain proteins are inherently unstable and undergo "default" degradation by the 20S proteasomes. Puzzlingly, proteins are by large subjected to both degradation pathways. Proteins with unstructured regions have been found to be substrates of the 20S proteasomes in vitro and, therefore, unstructured regions may serve as signals for protein degradation "by default" in the cell. The literature is loaded with examples where engagement of a protein into larger complexes increases protein stability, possibly by escaping degradation "by default". Our model suggests that formation of protein complexes masks the unstructured regions, making them inaccessible to the 20S proteasomes. This model not only provides molecular explanations for a recent theoretical "cooperative stability" principle, but also provokes new predictions and explanations in the field of protein regulation and functionality.     

New crystal forms and low resolution structure analysis of 20S proteasomes from bovine liver

20S proteasomes from higher eukaryotes have immunological functions rather than those from archibacteria or yeast. To clarify the mechanism of the sorting and production of antigen-presenting peptides, it is important and worthwhile to determine the structure of mammalian proteasomes using a third generation synchrotron radiation source. Here we report new crystal forms of 20S proteasomes from bovine liver and preliminary structure analysis of them. The crystals belong to the same space group but have different cell dimensions. One crystal (form I) belongs to space group P2(1)2(1)2(1) with unit cell dimensions of a = 124.8, b =197.4, c =323.8 A, and diffracts to 3.0 A resolution. The other crystal (form II) belongs to the same space group with a =115.1, b =205.6, c =316. 0 A, and diffracts to 4.0 A resolution. The diffraction data for the form I crystal provided an interpretable electron density map for presenting the structural differences from yeast proteasomes.     

The subunit structure and dynamics of the 20S proteasome in chicken skeletal muscle

We have succeeded in purifying the 20S core proteasome particle from less than 1 g of skeletal muscle in a rapid process involving two chromatographic steps. The individual subunits were readily resolved by two-dimensional PAGE, and the identities of each of the 14 subunits were assigned by a combination of peptide mass fingerprinting and MS/MS/de novo sequencing. To assess the dynamics of proteasome biogenesis, chicks were fed a diet containing stable isotope-labeled valine, and the rate of incorporation of label into valine-containing peptides derived from each subunit was assessed by mass spectrometric analysis after two-dimensional separation. Peptides containing multiple valine residues from the 20S proteasome and other soluble muscle proteins were analyzed to yield the relative isotope abundance of the precursor pool, a piece of information that is essential for calculation of turnover parameters. The rates of synthesis of each subunit are rather similar, although there is evidence for high turnover subunits in both the alpha (nonproteolytic) and beta (proteolytic) rings. The variability in synthesis rate for the different subunits is consistent with a model in which some subunits are produced in excess, whereas others may be the rate-limiting factor in the concentration of 20S subunits in the cell. The ability to measure turnover rates of proteins on a proteome-wide scale in protein assemblies and in a complex organism provides a new dimension to the understanding of the dynamic proteome.     

Isolation and characterization of two 20S proteasomes from the endoplasmic reticulum of rat liver microsomes.

Two new forms of proteasomes, designated as the endoplasmic reticulum (ER) membrane-associated proteasome (ERa proteasome) and ER membrane-bound proteasome (ERb proteasome), were purified to homogeneity from 0.0125 and 2.5% sodium cholate extracts, respectively, of a rat liver microsomal fraction. SDS-PAGE analysis revealed that the purified ERa and ERb proteasomes were composed of multiple subunits similar to the cytosolic 20S proteasome. However, electrophoretic, structural and immunochemical differences between the ERa, ERb and cytosolic 20S proteasomes were observed on native PAGE, two-dimensional (2D) PAGE, and immunoblot analyses. Purification of ERb from a 2.5% sodium cholate extract of the trypsin-treated microsomal fraction yielded a trypsin-modified form of ERb (tERb), which lacked the C2 subunit at least. On the other hand, no ERa proteasome was obtained from the 0.0125% sodium cholate extract of the trypsin-treated microsomes, suggesting that ERa and ERb are ER membrane-associated and -bound proteasomes, respectively. The ERa, ERb, and cytosolic 20S proteasomes exhibited similar specificities as to peptide hydrolyzing activity, although differences in their activities were noted in the presence of SDS and phospholipid. With respect to the proteolysis of protein substrates, only the ERb proteasome cleaved beta-casein, and it also degraded reduced and carboxymethylated lysozyme considerably faster than the cytosolic 20S and ERa proteasomes. Collectively our results suggest that the ERa and ERb proteasomes may play roles in intracellular proteolysis distinct from that of the cytosolic 20S proteasome.     

Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes

The 20S proteasome is a catalytic core of the 26S proteasome, a central enzyme in the degradation of ubiquitin-conjugated proteins. It is composed of 14 distinct gene products that form four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). It is reported that the biogenesis of mammalian 20S proteasomes is assisted by proteasome-specific chaperones, named PAC1, PAC2, and hUmp1, but the details are still unknown. Here, we report the identification of a chaperone, designated PAC3, as a component of alpha rings. Although it can intrinsically bind directly to both alpha and beta subunits, PAC3 dissociates before the formation of half-proteasomes, a process coupled with the recruitment of beta subunits and hUmp1. Knockdown of PAC3 impaired alpha ring formation. Further, PAC1/2/3 triple knockdown resulted in the accumulation of disorganized half-proteasomes that are incompetent for dimerization. Our results describe a cooperative system of multiple chaperones involved in the correct assembly of mammalian 20S proteasomes.     

20 S proteasomes are imported as precursor complexes into the nucleus of yeast

The mechanism by which yeast 20 S proteasomes are imported into the nucleus is still unresolved. Here, we provide the first evidence that 20 S proteasomes are imported as precursor complexes into the nucleus. By using the srp1-49 mutant which is deficient in nuclear import of cargos with classical nuclear localization sequences (cNLS), we show that proteasome precursor complexes associate with importin/karyopherin alphabeta, the cNLS receptor, and that they accumulate inside the cytoplasm. Reconstitution assays revealed that only precursor complexes are targeted to the nuclear envelope (NE) by karyopherin alphabeta. In support, the green fluorescent protein (GFP)-labelled maturation factor Ump1, marking precursor complexes, mainly localizes to the nucleus and around the NE. Our data suggest that nuclear 20 S proteasomes are finally matured inside the nucleus.     

Substrate size selectivity of 20S proteasomes: analysis with variable-sized synthetic substrates

Proteasomes are tubular complexes with proteolytic activities on their lumenal surfaces so that large substrates should be sterically hindered from reaching the catalytic sites. Here we examine effects of substrate size on rates of cleavage by 20S proteasomes of Methanosarcina thermophila. Synthetic chromogenic substrates of variable size were prepared by linking a constant substrate group (Ala-Ala-Phe-p-nitroanilide) to a linear polymer (methoxypolyethylene glycol) with variable chain length. The smallest macromolecular substrates were cleaved more efficiently than free tripeptide substrate, and cleavage of macromolecular substrates was saturable, whereas cleavage of free tripeptide substrate was not, indicating mechanistic differences between the cleavage of large and small substrates. Rates of macromolecular substrate cleavage decreased progressively up to 10-fold as the size of the polymeric component of substrates increased. Macromolecular synthetic substrates appear to be better models of proteasome action on natural protein substrates and demonstrate substrate size selectivity of proteasomes.     

The human 26 S and 20 S proteasomes generate overlapping but different sets of peptide fragments from a model protein substrate

Intracellular protein degradation is a major source of short antigenic peptides that can be presented on the cell surface in the context of major histocompatibility class I molecules for recognition by cytotoxic T lymphocytes. The capacity of the most important cytosolic protease, the 20 S proteasome, to generate peptide fragments with an average length of 7-8 amino acid residues has been thoroughly investigated. It has been shown that the cleavage products are not randomly generated, but originate from the commitment of the catalytically active subunits to complex recognition motifs in the primary amino acid sequence. The role of the even larger 26 S proteasome is less well defined, however. It has been demonstrated that the 26 S proteasome can bind and degrade ubiquitin-tagged proteins and minigene translation products in vivo and in vitro, but the nature of the degradation products remains elusive. In this study, we present the first analysis of cleavage products from in vitro digestion of the unmodified model substrate beta-casein with both the 26 S and 20 S proteasome. The data we obtained show that 26 S and 20 S proteasomes generate overlapping, but at the same time substantially different, sets of fragments by following very similar instructions.     

The pore of activated 20S proteasomes has an ordered 7-fold symmetric conformation

The 20S proteasome is a large multisubunit assembly that performs most of the intracellular non-lysosomal proteolysis of eukaryotes. Substrates access the proteasome active sites, which are sequestered in the interior of the barrel-shaped structure, through pores that are opened by binding of activator complexes. The crystal structure of yeast proteasome in complex with an 11S activator suggested that activation results from disordering of the proteasome gate residues. Here we report further analysis of this structure, which demonstrates that, in contrast to earlier models, the activated proteasome adopts an ordered 7-fold symmetric pore conformation that is stabilized by interactions formed by a cluster of highly conserved proteasome residues (Tyr8, Asp9, Pro17 and Tyr26). One non-canonical cluster, which appears to be mandated by the requirement that eukaryotic proteasomes also form an ordered closed conformation, explains all deviations from perfect conservation of these residues. We also demonstrate the importance of these conserved residues for proteolysis by an archaeal proteasome. Evolutionary considerations suggest that other activators might induce the same open proteasome conformation as seen with the 11S activator.