Structural features of archaebacterial and eukaryotic proteasomes

The 26S proteasome is the central protease of the ubiquitin-dependent pathway of protein degradation. The molecule has a molecular mass of approximately 2000 kD and has a highly conserved structure in eukaryotes. The 26S proteasome is formed by a barrel-shaped 20S core complex and two polar 19S complexes. The 20S complex has C2 symmetry and is formed by four seven-membered rings of which the outer rings (-type subunits) are rotated by 25.7° relative to the inner rings while the inner rings (-type subunits) are in register. From a comparison of the activity and regulation of the 26S and 20S particles it can be deduced that the 20S particle contains the protease activity while the 19S complex contains isopeptidase, ATPase and protein unfolding activities. In this article we describe the structures of various proteasome complexes as determined by electron microscopy and discuss structural implications of their subunit sequences.     

Eubacterial proteasomes

Proteasomes are large, multisubunit proteases with highly conserved structures. The 26S proteasome of eukaryotes is an ATP-dependent enzyme of about 2 MDa, which acts as the central protease of the ubiquitin-dependent pathway of protein degradation. The core of the 26S complex is formed by the 20S proteasome, an ATP-independent, barrel-shaped protease of about 700 kDa, which has also been detected in archaebacteria and, more recently, in eubacteria. Currently, the distribution of 20S proteasomes in eubacteria appears limited to the actinomycetes, while most other eubacteria contain a related complex of simpler structure.     

Electron microscopy of 26 S complex containing 20 S proteasome.

A high molecular weight protease complex (26 S complex) involved in the intracellular protein degradation of ubiquitinated proteins was purified from rat liver and studied by electron microscopy. The most prevalent molecular species with best preserved symmetrical morphology had two large rectangular terminal structures attached to a thinner central one having four protein layers. We concluded that they were the closest representation of the 26 S complex so far reported. The central structure was identified as 20 S proteasome and the terminal one as recognition units for ubiquitinated proteins.     

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.     

GFP-labelling of 26S proteasomes in living yeast: insight into proteasomal functions at the nuclear envelope/rough ER

26S proteasomes are multisubunit protease complexes that play the central role in the ubiquitin-dependent protein degradation pathway. The proteolytically active core is formed by the 20S proteasome. Regulatory subunits, principally the 19S cap complex, confer the specificity towards ubiquitinated substrates and an ATP-dependence on proteolysis. Green fluorescence protein (GFP)-tagged versions of either an -subunit of the 20S core or an ATPase subunit of the 19S cap complex were functionally incorporated into the protease complex, thus allowing to monitor the subcellular distribution of 26S proteasomes in living yeast. Our localization studies suggest that proteasomal proteolysis mainly occurs at the nuclear envelope (NE)/rough ER. Implications of proteasomal functions at the NE/rough ER are discussed in the context of published work on ER degradation and with regard to possible targeting mechanisms.     

Quaternary structure of the ATPase complex of human 26S proteasomes determined by chemical cross-linking

The 26S proteasome is the major protease responsible for nonlysosomal protein degradation in eukaryotic cells. The enzyme is composed of two subparticles: the 20S proteasome, and a 19S regulatory particle (PA700) which binds to the ends of the 20S proteasome cylinder and accounts for ATP dependence and substrate specificity. Among the approximately 18 subunits of PA700 regulator, six are ATPases. The ATPases presumably recognize, unfold, and translocate substrates into the interior of the 26S proteasome. It is generally believed that the ATPases form a hexameric ring. By means of chemical cross-linking, immunoprecipitation, and blotting, we have determined that the ATPases are organized in the order S6-S6'-S10b-S8-S4-S7. Additionally, we found cross-links between the ATPase S10b and the 20S proteasome subunit alpha6. Together with the previously known interaction between S8 and alpha1 and between S4 and alpha7, these data establish the relative orientations of ATPases with respect to the 20S proteasome.     

20S proteasome biogenesis

26S proteasomes are multi-subunit protease complexes responsible for the turnover of short-lived proteins. Proteasomal degradation starts with the autocatalytic maturation of the 20S core particle. Here, we summarize different models of proteasome assembly. 20S proteasomes are assembled as precursor complexes containing alpha and unprocessed beta subunits. The propeptides of the beta subunits are thought to prevent premature conversion of the precursor complexes into matured particles and are needed for efficient beta subunit incorporation. The complex biogenesis is tightly regulated which requires additional components such as the maturation factor Ump1/POMP, an ubiquitous protein in eukaryotic cells. Ump1/POMP is associated with precursor intermediates and degraded upon final maturation. Mammalian proteasomes are localized all over the cell, while yeast proteasomes mainly localize to the nuclear envelope/endoplasmic reticulum (ER) membrane network. The major localization of yeast proteasomes may point to the subcellular place of proteasome biogenesis.     

Structural features of the 26S proteasome complex isolated from rat testis and sperm tail

We have previously cloned a cDNA encoding TBP-1, a protein present in the rat spermatid manchette and outer dense fibers of the developing sperm. TBP-1 contains a heptad repeat of six-leucine zipper fingers at the amino terminus and highly conserved ATPase and DNA/RNA helicase motifs toward the carboxyl terminus. TBP-1 is one of the 20 subunits forming the 19S regulatory complex of the 26S proteasome, an ATP-dependent multisubunit protease found in most eukaryotic cells. We now report the isolation of the 26S proteasome from rat testis and sperm tail and its visualization by whole-mount electron microscopy using negative staining. The 26S proteasome from rat testis was fractionated by Sephacryl S-400/Mono-Q chromatography using homogenates suspended in a 10% glycerol-supplemented buffer. Chromatographic fractions were analyzed by immunoblotting using a specific anti-TBP-1 serum. During the purification of Sak57, a keratin filament present in outer dense fibers from epididymal sperm, we detected a substantial amount of 26S proteasomes. Intact 26S proteasomes from rat testis display a rod-shaped particles about 45 nm in length and 11-17 nm in diameter. Each particle consists of a 20S barrel-shaped component formed by four rings (alphabetabetaalpha), capped by two polar 19S regulatory complexes, each identified by an element known as the "Chinese dragon head motif". TBP-1 is an ATPase-containing subunit of the 19S regulatory cap. Rat sperm preparations displayed both dissociated 26S proteasomes and Sak57 filaments. We hypothesize that 26S proteasomes in the perinuclear-arranged manchette are in a suitable location for recognition, sequestration, and degradation of accumulating ubiquitin-conjugated somatic and transient testis-specific histones during spermiogenesis. In the sperm tail, the 26S proteasome may have a role in the remodeling of the outer dense fibers and other tail components during epididymal transit.     

Dissecting protein‐protein interactions in proteasome assembly: Implication to its self‐assembly

The 26S proteasome is a multi‐catalytic ATP‐dependent protease complex that recognizes and cleaves damaged or misfolded proteins to maintain cellular homeostasis. The 26S subunit consists of 20S core and 19S regulatory particles. 20S core particle consists of a stack of heptameric alpha and beta subunits. To elucidate the structure‐function relationship, we have dissected protein‐protein interfaces of 20S core particle and analyzed structural and physiochemical properties of intra‐alpha, intra‐beta, inter‐beta, and alpha‐beta interfaces. Furthermore, we have studied the evolutionary conservation of 20S core particle. We find the size of intra‐alpha interfaces is significantly larger and is more hydrophobic compared with other interfaces. Inter‐beta interfaces are well packed, more polar, and have higher salt‐bridge density than other interfaces. In proteasome assembly, residues in beta subunits are better conserved than alpha subunits, while multi‐interface residues are the most conserved. Among all the residues at the interfaces of both alpha and beta subunits, Gly is highly conserved. The largest size of intra‐alpha interfaces complies with the hypothesis that large interfaces form first during the 20S assembly. The tight packing of inter‐beta interfaces makes the core particle impenetrable from outer wall of the cylinder. Comparing the three domains, eukaryotes have large and well‐packed interfaces followed by archaea and bacteria. Our findings provide a structural basis of assembly of 20S core particle in all the three domains of life.     

Affinity Purification of the Arabidopsis 26 S Proteasome Reveals a Diverse Array of Plant Proteolytic Complexes

Selective proteolysis in plants is largely mediated by the ubiquitin (Ub)/proteasome system in which substrates, marked by the covalent attachment of Ub, are degraded by the 26 S proteasome. The 26 S proteasome is composed of two subparticles, the 20 S core protease (CP) that compartmentalizes the protease active sites and the 19 S regulatory particle that recognizes and translocates appropriate substrates into the CP lumen for breakdown. Here, we describe an affinity method to rapidly purify epitope-tagged 26 S proteasomes intact from Arabidopsis thaliana. In-depth mass spectrometric analyses of preparations generated from young seedlings confirmed that the 2.5-MDa CP-regulatory particle complex is actually a heterogeneous set of particles assembled with paralogous pairs for most subunits. A number of these subunits are modified post-translationally by proteolytic processing, acetylation, and/or ubiquitylation. Several proteasome-associated proteins were also identified that likely assist in complex assembly and regulation. In addition, we detected a particle consisting of the CP capped by the single subunit PA200 activator that may be involved in Ub-independent protein breakdown. Taken together, it appears that a diverse and highly dynamic population of proteasomes is assembled in plants, which may expand the target specificity and functions of intracellular proteolysis.     

Structure of the human 26S proteasome: subunit radial displacements open the gate into the proteolytic core

The 26S proteasome plays an essential role in regulating many cellular processes by the degradation of proteins targeted for breakdown by ubiquitin conjugation. The 26S complex is formed from the 20S core, which contains the proteolytic active sites, and 19S regulatory complexes, which bind to the 20S core to activate it and confer specificity for ubiquitinated protein substrates. We have determined the structure of the human 26S proteasome by electron microscopy and single particle analysis. In our reconstructions the crystallographic structure of each of the subunits of the 20S core can be unambiguously docked by direct recognition of each of their densities. Our results show for the first time that binding of the 19S regulatory particle results in the radial displacement of the adjacent subunits of the 20S core leading to opening of a wide channel into the proteolytic chamber. The analysis of a proteasome complex formed from one 20S core with a single 19S regulatory particle attached serve as control to our observations. We suggest locations for some of the 19S regulatory particle subunits.     

The 26S proteasome: subunits and functions

The 26S proteasome is an eukaryotic ATP-dependent, dumbbell-shaped protease complex with a molecular mass of approximately 2000 kDa. It consists of a central 20S proteasome, functioning as a catalytic machine, and two large V-shaped terminal modules, having possible regulatory roles, composed of multiple subunits of 25–110 kDa attached to the central portion in opposite orientations. The primary structures of all the subunits of mammalian and yeast 20S proteasomes have been determined by recombinant DNA techniques, but structural analyses of the regulatory subunits of the 26S proteasome are still in progress. The regulatory subunits are classified into two subgroups, a subgroup of at least 6 ATPases that constitute a unique multi-gene family encoding homologous polypeptides conserved during evolution and a subgroup of approximately 15 non-ATPase subunits, most of which are structurally unrelated to each other.     

Multiple forms of the 20 S multicatalytic and the 26 S ubiquitin/ATP-dependent proteases from rabbit reticulocyte lysate.

We have used native gel electrophoresis followed by fluorogenic peptide overlay to identify multiple forms of rabbit reticulocyte multicatalytic protease (MCP) or 20 S protease, and two forms of rabbit 26 S ubiquitin/ATP-dependent protease. An abundant, fast-migrating 20 S complex (20 SF) possesses modest ability to hydrolyze the fluorogenic peptide succinyl-Leu-Leu-Val-Tyr-4-methyl-coumaryl-7-amide. In contrast, two minor, slower migrating species cleave the peptide at high rates. A unique 30-kDa polypeptide is associated with one of the active MCPs, and a 160-kDa subunit is associated with the other. Two electrophoretically distinct 26 S proteases can also be isolated from rabbit reticulocyte lysate. The faster migrating form, 26 SF, is more resistant to inactivation by ATP depletion. Despite the differential response to nucleotides and the distinctive electrophoretic mobilities of 26 SF and 26 SS, we have not identified any subunit differences between the two enzymes. In addition to active 26 S proteases, we have discovered and purified a proteolytically inactive particle that contains subunits characteristic of the 26 S protease (e.g. molecular masses between 30 and 110 kDa). Incubation of this protein complex with purified MCP and ATP results in the formation of the 26 S proteases.     

Aggregated and monomeric alpha-synuclein bind to the S6' proteasomal protein and inhibit proteasomal function

The accumulation of aggregated alpha-synuclein is thought to contribute to the pathophysiology of Parkinson's disease, but the mechanism of toxicity is poorly understood. Recent studies suggest that aggregated proteins cause toxicity by inhibiting the ubiquitin-dependent proteasomal system. In the present study, we explore how alpha-synuclein interacts with the proteasome. The proteasome exists as a 26 S and a 20 S species. The 26 S proteasome is composed of the 19 S cap and the 20 S core. Aggregated alpha-synuclein strongly inhibited the function of the 26 S proteasome. The IC(50) of aggregated alpha-synuclein for ubiquitin-independent 26 S proteasomal activity was 1 nm. Aggregated alpha-synuclein also inhibited 26 S ubiquitin-dependent proteasomal activity at a dose of 500 nm. In contrast, the IC(50) of aggregated alpha-synuclein for 20 S proteasomal activity was > 1 microm. This suggests that aggregated alpha-synuclein selectively interacts with the 19 S cap. Monomeric alpha-synuclein also inhibited proteasomal activity but with lower affinity and less potency. Recombinant monomeric alpha-synuclein inhibited the activity of the 20 S proteasomal core with an IC(50) > 10 microm, exhibited no inhibition of 26 S ubiquitin-dependent proteasomal activity at doses up to 5 microm, and exhibited only partial inhibition (50%) of the 26 S ubiquitin-independent proteasomal activity at doses up to 10 mm. Binding studies demonstrate that both aggregated and monomeric alpha-synuclein selectively bind to the proteasomal protein S6', a subunit of the 19 S cap. These studies suggest that proteasomal inhibition by aggregated alpha-synuclein could be mediated by interaction with S6'.     

Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin-ligated proteins by closely linked mechanisms.

It is known that two types of high-molecular-mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP-dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney. In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 x g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular-sieve chromatography on a Biogel A-1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density-gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21-31 kDa and 13-15 protein components ranging in molecular mass over 35-110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I-labeled lysozyme-ubiquitin conjugates in an ATP-dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP-dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy-dependent, ubiquitin-mediated protein degradation.     

ASK1 Negatively Regulates the 26 S Proteasome

The 26 S proteasome, composed of the 20 S core and 19 S regulatory particle, plays a central role in ubiquitin-dependent proteolysis. Disruption of this process contributes to the pathogenesis of the various diseases; however, the mechanisms underlying the regulation of 26 S proteasome activity remain elusive. Here, cell culture experiments and in vitro assays demonstrated that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAPK kinase kinase family, negatively regulated 26 S proteasome activity. Immunoprecipitation/Western blot analyses revealed that ASK1 did not interact with 20 S catalytic core but did interact with ATPases making up the 19 S particle, which is responsible for recognizing polyubiquitinated proteins, unfolding them, and translocating them into the 20 S catalytic core in an ATP-dependent process. Importantly, ASK1 phosphorylated Rpt5, an AAA ATPase of the 19 S proteasome, and inhibited its ATPase activity, an effect that may underlie the ability of ASK1 to inhibit 26 S proteasome activity. The current findings point to a novel role for ASK1 in the regulation of 26 S proteasome and offer new strategies for treating human diseases caused by proteasome malfunction.     

Differential roles of the COOH termini of AAA subunits of PA700 (19 S regulator) in asymmetric assembly and activation of the 26 S proteasome

The 26 S proteasome is an energy-dependent protease that degrades proteins modified with polyubiquitin chains. It is assembled from two multi-protein subcomplexes: a protease (20 S proteasome) and an ATPase regulatory complex (PA700 or 19 S regulatory particle) that contains six different AAA family subunits (Rpt1 to -6). Here we show that binding of PA700 to the 20 S proteasome is mediated by the COOH termini of two (Rpt2 and Rpt5) of the six Rpt subunits that constitute the interaction surface between the subcomplexes. COOH-terminal peptides of either Rpt2 or Rpt5 bind to the 20 S proteasome and activate hydrolysis of short peptide substrates. Simultaneous binding of both COOH-terminal peptides had additive effects on peptide substrate hydrolysis, suggesting that they bind to distinct sites on the proteasome. In contrast, only the Rpt5 peptide activated hydrolysis of protein substrates. Nevertheless, the COOH-terminal peptide of Rpt2 greatly enhanced this effect, suggesting that proteasome activation is a multistate process. Rpt2 and Rpt5 COOH-terminal peptides cross-linked to different but specific subunits of the 20 S proteasome. These results reveal critical roles of COOH termini of Rpt subunits of PA700 in the assembly and activation of eukaryotic 26 S proteasome. Moreover, they support a model in which Rpt subunits bind to dedicated sites on the proteasome and play specific, nonequivalent roles in the asymmetric assembly and activation of the 26 S proteasome.     

Initiation of Cyclin B Degradation by the 26S Proteasome upon Egg Activation

Immediately before the transition from metaphase to anaphase, the protein kinase activity of maturation or M-phase promoting factor (MPF) is inactivated by a mechanism that involves the degradation of its regulatory subunit, cyclin B. The availability of biologically active goldfish cyclin B produced in Escherichia coli and purified goldfish proteasomes (a nonlysosomal large protease) has allowed the role of proteasomes in the regulation of cyclin degradation to be examined for the first time. The 26S, but not the 20S proteasome, digested recombinant 49-kD cyclin B at lysine 57 (K57), producing a 42-kD truncated form. The 42-kD cyclin was also produced by the digestion of native cyclin B forming a complex with cdc2, a catalytic subunit of MPF, and a fragment transiently appeared during cyclin degradation when eggs were released from metaphase II arrest by egg activation. Mutant cyclin at K57 was resistant to both digestion by the 26S proteasome and degradation at metaphase/anaphase transition in Xenopus egg extracts. The results of this study indicate that the destruction of cyclin B is initiated by the ATP-dependent and ubiquitin-independent proteolytic activity of 26S proteasome through the first cutting in the NH₂ terminus of cyclin (at K57 in the case of goldfish cyclin B). We also surmise that this cut allows the cyclin to be ubiquitinated for further destruction by ubiquitin-dependent activity of the 26S proteasome that leads to MPF inactivation.     

Assembly of the 26 S complex that degrades proteins ligated to ubiquitin is accompanied by the formation of ATPase activity

In the ubiquitin pathway for intracellular protein breakdown, proteins ligated to ubiquitin are degraded by a large (26 S) ATP-dependent protease complex. It was found previously that the 26 S complex is assembled from three different enzyme components by a process that requires MgATP. In addition, MgATP is also required for the continued action of the 26 S complex in the breakdown of ubiquitin-protein conjugates. In the present study we have tried to gain some insight into the mode of action of ATP by following ATP hydrolysis by the 26 S complex and its three components. It was found that none of the three unassembled components had significant ATPase activity, but such activity appeared following their entry into the 26 S complex. The presence of all three components and of MgATP was required for the formation of complex-associated ATPase activity. GTP and UTP cannot replace ATP for complex assembly, but these nucleotides can substitute for ATP in the stimulation of the conjugate-degrading activity of the 26 S complex. Unlabeled GTP and UTP inhibit the hydrolysis of [gamma-32P] ATP by complex-associated ATPase, indicating that this activity is related to the latter site of ATP action in this system.     

Sem1p is a novel subunit of the 26 S proteasome from Saccharomyces cerevisiae

The 26 S proteasome, which catalyzes degradation of polyubiquitinated proteins, is composed of the 20 S proteasome and the 19 S regulatory particle (RP). The RP is composed of the lid and base subcomplexes and regulates the catalytic activity of the 20 S proteasome. In this study, we carried out affinity purification of the lid and base subcomplexes from the tagged strains of Saccharomyces cerevisiae, and we found that the lid contains a small molecular mass protein, Sem1. The Sem1 protein binds with the 26 S proteasome isolated from a mutant with deletion of SEM1 but not with the 26 S proteasome from the wild type. The lid lacking Sem1 is unstable at a high salt concentration. The 19 S RP was immunoprecipitated together with Sem1 by immunoprecipitation using hemagglutinin epitope-tagged Sem1 as bait. Degradation of polyubiquitinated proteins in vivo or in vitro is impaired in the Sem1-deficient 26 S proteasome. In addition, genetic interaction between SEM1 and RPN10 was detected. The human Sem1 homologue hDSS1 was found to be a functional homologue of Sem1 and capable of interacting with the human 26 S proteasome. The results suggest that Sem1, possibly hDSS1, is a novel subunit of the 26 S proteasome and plays a role in ubiquitin-dependent proteolysis.