Atomic force microscopy reveals two conformations of the 20 S proteasome from fission yeast

The proteasome is a major cytosolic proteolytic complex, indispensable in eukaryotic cells. The barrel-shaped core of this enzyme, the 20 S proteasome, is built from 28 subunits forming four stacked rings. The two inner beta-rings harbor active centers, whereas the two outer alpha-rings play a structural role. Crystal structure of the yeast 20 S particle showed that the entrance to the central channel was sealed. Because of this result, the path of substrates into the catalytic chamber has remained enigmatic. We have used tapping mode atomic force microscopy (AFM) in liquid to address the dynamic aspects of the 20 S proteasomes from fission yeast. We present here evidence that, when observed with AFM, the proteasome particles in top view position have either open or closed entrance to the central channel. The preferred conformation depends on the ligands present. Apparently, the addition of a substrate to the uninhibited proteasome shifts the equilibrium toward the open conformation. These results shed new light on the possible path of the substrate into the proteolytic chamber.     

Characterization of the 20S proteasome of the lepidopteran,Spodoptera frugiperda

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.     

Recombinant proteasomal alpha-type subunits exhibit endoribonuclease activity

26S proteasome is a multi-subunit protein complex that consists of the regulatory 19S and the catalytic 20S subcomplexes. The major cellular function of the proteasome is protein degradation. It has been found recently that the 20S particle, besides its proteolytic activity, also possesses endoribonuclease activity. The latter is mediated by two alpha-type subunits (alpha1 and alpha5). In this report we have analyzed the remaining alpha-type subunits for their ability to hydrolyze RNA. We found that all of the recombinant subunits tested exhibited endoribonuclease activity which depended on the origin of RNA and the presence of bivalent ions in the reaction. These results indicate that the endoribonuclease activity of proteasomes may play an important role in cellular metabolism of RNA.     

Proteasome subunits are regulated and expressed in comparable concentrations as a functional cluster

The proteasome is the main proteolytic enzyme that functions in the ubiquitin-proteasome system. The 26S proteasome has multi-subunit protease complexes consisting of 20S subunits composed of four seven-numbered rings with two outer rings containing α subunits and two central rings composed of β subunits, and 19S caps of 6 ATPase and 11 non-ATPase subunits; however, it is unclear how these subunits are regulated and the 26S proteasomes assembled. To verify whether each subunit’s mRNA expression is associated with the mRNA expression of other proteasome subunits, we carried out expression analysis of 34 proteasome subunits mRNA on peripheral blood from 75 subjects. The expression of proteasome subunits mRNA was comparable in each individual of the studied population and the mRNA expression has been investigated in each 20S or 19S proteasome. Our results suggest that each type of subunit is regulated by respectively common factors, and that the 20S and 19S proteasomes are regulated by different systems.     

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.     

Interaction of human erythrocyte multicatalytic proteinase with polycations

The multicatalytic proteinase from human erythrocytes (macropain, proteasome) is a large enzyme composed of at least six distinct subunits ranging in molecular masses from 20 to 30 kDa. As its name implies, this proteinase appears to contain multiple catalytic sites with differing specificities toward peptide substrates. Several polycationic substances, including polylysines, polyarginine, protamine and histone H1 markedly stimulated caseinolytic activity of the proteinase. Activation was instantaneous, and involved increasing the Vmax of the proteinase for casein. Prolonged preincubation with polylysine at 37 degrees C resulted in autolytic inactivation of the proteinase. The polylysine concentrations required for half-maximal activation or autolytic inactivation were the same. A 23 kDa subunit of the proteinase disappeared at the same rate as loss of catalytic activity, and with the same pH dependence and polylysine concentration dependence. These results suggest that polylysine perturbs the structure of the multicatalytic proteinase, resulting in increased catalytic activity toward substrates; and, with prolonged exposure, allowing autoproteolytic inactivation to occur. The 23 kDa subunit appeared to be required for expression of caseinolytic activity, and may therefore be a catalytic subunit of the complex having activity against casein.     

Recombinant proteasome alpha-type subunits exhibit endoribonuclease activity

26S proteasome is a multisubunit protein complex that consists of 19S regulatory and 20S catalytic subcomplexes. The primary proteasome cellular function is protein degradation. It has recently been found that, in addition to its proteolytic activities, the 20S particle also displays endoribonuclease activity mediated by two alpha-type subunits, α1 and α5. In this report, we have analyzed other alpha-type subunits for their ability to hydrolyze RNA. We have found that all of the recombinant subunits tested (α1, α2, α3, α4, α5, α7) exhibited endoribonuclease activity that depends on the origin of RNA and the presence of bivalent ions in the reaction. These results indicate that the endoribonuclease activity of proteasomes may play an important role in cellular RNA metabolism.     

Affinity labeling of the proteasome by a belactosin A derived inhibitor

Belactosin A is a potent proteasome inhibitor isolated from Streptomyces metabolites. Here we show that a hydrophobic belactosin A derivative, dansyl-KF33955, can covalently, and specifically, affinity label the catalytic subunits of the 26S proteasome, which consists of the 20S protein degrading core particle and the 19S regulatory particles. The labeling of catalytic subunits proceeds faster in intact proteasomes in vivo than in isolated 20S core particles. These data suggest that the 19S regulatory particle may facilitate entry of the inhibitor into the 20S core particle. This cell-permeable chemical probe is an excellent tool with which to study the interactions of this proteasome inhibitor with proteasomes in intact cells.     

The proteasome-dependent proteolytic system

The 20S proteasome is an intriguingly large complex that acts as a proteolytic catalytic machine. Accumulating evidence indicates the existence of multiple factors capable of regulating the proteasome function. They are classified into two different categories, one type of regulator is PA700 or PA28 that is reversibly associated with the 20S proteasome to form enzymatically active proteasomes and the other type including a 300-kDa modulator and PI31 indirectly influences proteasome activity perhaps by promoting or suppressing the assembly of the 20S proteasome with PA700 or PA28. Thus, there have been documented two types of proteasomes composed of a core catalytic proteasome and a pair of symmetrically disposed PA700 or PA28 regulatory particle. Moreover, the recently-identified proteasome containing both PA28 and PA700 appears to play a significant role in the ATP-dependent proteolytic pathway in cells, as can the 26S proteasome which is known as a eukaryotic ATP-dependent protease.     

Proteasomes of the yeastS. cerevisiae: genes,structure and functions

Proteasomes are large multicatalytic protease complexes which fulfil central functions in major intracellular proteolytic pathways of the eukaryotic cell. 20S proteasomes are 700 kDa cylindrically shaped particles, found in the cytoplasm and the nucleus of all eukaryotes. They are composed of a pool of 14 different subunits (MW 22–25 kDa) arranged in a stack of 4 rings with 7-fold symmetry. In the yeastSaccharomyces cerevisiae a complete set of 14 genes coding for 20S proteasome subunits have been cloned and sequenced. 26S proteasomes are even larger proteinase complexes (about 1700 kDa) which degrade ubiquitinylated proteins in an ATP-dependent fashionin vitro. The 26S proteasome is build up from the 20S proteasome as core particle and two additional 19S complexes at both ends of the 20S cylinder. Recently existence of a 26S proteasome in yeast has been demonstrated. Several 26S proteasome specific genes have been cloned and sequenced. They share similarity with a novel defined family of ATPases. 20S and 26S proteasomes are essential for functioning of the eukaryotic cell. Chromosomal deletion of 20S and 26S proteasomal genes in the yeastS. cerevisiae caused lethality of the cell. Thein vivo functions of proteasomes in major proteolytic pathways have been demonstrated by the use of 20S and 26S proteasomal mutants. Proteasomes are needed for stress dependent and ubiquitin mediated proteolysis. They are involved in the degradation of short-lived and regulatory proteins. Proteasomes are important for cell differentiation and adaptation to environmental changes. Proteasomes have also been shown to function in the control of the cell cycle.     

Substrate access and processing by the 20S proteasome core particle

Intracellular proteolysis is an essential process. In eukaryotes, most proteins in the cytosol and nucleus are degraded by the ubiquitin (Ub)-proteasome pathway. A major component within this system is the 26S proteasome, a 2.5MDa molecular machine, built from more than 31 different subunits. This complex is formed by a cylinder-shaped multimeric complex referred to as the proteolytic 20S proteasome (core particle, CP) capped at each end by another multimeric component called the 19S complex (regulatory particle, RP) or PA700. Structure, assembly and enzymatic mechanism have been elucidated only for the CP, whereas the organization of the RP is less well understood. The CP is composed of 28 subunits, which are arranged as an alpha7beta7beta7alpha7-complex in four stacked rings. The interior of the free core particle, which harbors the active sites, is inaccessible for folded and unfolded substrates and represents a latent state. This inhibition is relieved upon binding of the RP to the CP by formation of the 26S proteasome holoenzyme. This review summarizes the current knowledge of the structural features of 20S proteasomes.     

Mammalian 26S proteasomes remain intact during protein degradation

It has been suggested that degradation of polyubiquitylated proteins is coupled to dissociation of 26S proteasomes. In contrast, using several independent types of experiments, we find that mammalian proteasomes can degrade polyubiquitylated proteins without disassembling. Thus, immobilized, (35)S-labeled 26S proteasomes degraded polyubiquitylated Sic1 and c-IAP1 without releasing any subunits. In addition, it is predicted that if 26S proteasomes dissociate into 20S proteasomes and regulatory complexes during a degradation cycle, the reassembly rate would be limiting at low proteasome concentrations. However, the rate with which each proteasome degraded polyubiquitylated Sic1 was independent of the proteasome concentration. Likewise, substrate-dependent dissociation of 26S proteasomes could not be detected by nondenaturing electrophoresis. Lastly, epoxomicin-inhibited 20S proteasomes can trap released regulatory complexes, forming inactive 26S proteasomes, but addition of epoxomicin-inhibited 20S proteasomes had no effect on the degradation of either polyubiquitylated Sic1 or UbcH10 by 26S proteasomes or of endogenous substrates in cell extracts.     

Decreased activity of the 20S proteasome in the brain white matter and gray matter of patients with multiple sclerosis

Carbonylated (oxidized) proteins are known to accumulate in the cerebral white matter (WM) and gray matter (GM) of patients with multiple sclerosis (MS). Although oxidative stress is necessary for carbonyl generation, it is the failure of the degradation systems that ultimately leads to the build-up of carbonylated proteins within tissues. In this study, we measured the activity of the 20S proteasome and other proteolytic systems in the cerebral WM and GM of 13 MS patients and 13 controls. We report that the activities of the three peptidases of the 20S proteasome (i.e. chymotrypsin-like, caspase-like and trypsin-like) in both MS-WM and MS-GM are greatly reduced. Interestingly, neither the amount of proteasome nor the levels of the catalytic subunits (β1, β2, and β5) are diminished in this disease. Proteins containing Lys-48 poly-ubiquitin also accumulate in MS tissues, indicating failure of the 26S proteasome as well. Levels of the regulatory caps 11S α and 19S are also lower in MS than in controls, suggesting that the activity of the more complex proteasomes may be reduced further. Finally, the activities of other proteases that might also remove oxidized proteins (calpain, cathepsin B, mitochondrial LonP) are not lessened in MS. Together, these studies suggest that direct inactivation of proteolytic centers in the 20S particle and/or the presence of specific inhibitors is the underlying cause of proteasomal dysfunction in MS.     

Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly.

Proteins targeted for degradation by the ubiquitin-proteasome system are degraded by the 26S proteasome. The core of this large protease is the 20S proteasome, a barrel-shaped structure made of a stack of four heptameric rings. Of the 14 different subunits that make up the yeast 20S proteasome, three have proteolytic active sites: Doa3/beta5, Pup1/beta2 and Pre3/beta1. Each of these subunits is synthesized with an N-terminal propeptide that is autocatalytically cleaved during particle assembly. We show here that the propeptides have both common and distinct functions in proteasome biogenesis. Unlike the Doa3 propeptide, which is crucial for proteasome assembly, the Pre3 and Pup1 propeptides are dispensable for cell viability and proteasome formation. However, mutants lacking these propeptide-encoding elements are defective for specific peptidase activities, are more sensitive to environmental stresses and have subtle defects in proteasome assembly. Unexpectedly, a critical function of the propeptide is the protection of the N-terminal catalytic threonine residue against Nalpha-acetylation. For all three propeptide-deleted subunits, activity of the affected catalytic center is fully restored when the Nat1-Ard1 Nalpha-acetyltransferase is mutated. In addition to delineating a novel function for proteasome propeptides, these data provide the first biochemical evidence for the postulated participation of the alpha-amino group in the proteasome catalytic mechanism.     

Proteasome structures affected by ionizing radiation

Exposure of cells to ionizing radiation slows the rate of degradation of substrates through the proteasome. Because the 26S proteasome degrades most short-lived cellular proteins, changes in its activity might significantly, and selectively, alter the life span of many signaling proteins and play a role in promoting the biological consequences of radiation exposure, such as cell cycle arrest, DNA repair, and apoptosis. Experiments were therefore undertaken to identify the radiation target that is associated with the proteasome. Regardless of whether they were irradiated before or after extraction and purification from human prostate cancer PC3 cells, 26S proteasomes remained intact but showed a rapid 30% to 50% dose-independent decrease in their three major enzymatic activities following exposure to 1 to 20 Gy. There was no effect on 20S proteasomes, suggesting that the radiation-sensitive target is located in the 19S cap of the 26S proteasome, rather than in the enzymatically active core. Because the base of the 19S cap contains an ATPase ring that mediates substrate unfolding, pore opening, and translocation of substrates into the catalytic chamber, we examined whether the ATPase activity of purified 26S proteasomes was affected. In fact, in vitro irradiation of proteasomes enhanced their ATPase activity. Furthermore, pretreatment with low concentrations of the free radical scavenger tempol was able to prevent both the radiation-induced decrease in proteolytic activity and the increase in ATP utilization, indicating that free radicals are mediators of these radiation-induced phenomena. Finally, we have shown that cell irradiation results in the accumulation of proteasome substrates: polyubiquitinated proteins and ornithine decarboxylase, indicating that the observed decrease in proteasome function is physiologically relevant.     

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.     

A proteasome assembly defect in rpn3 mutants is associated with Rpn11 instability and increased sensitivity to stress

Rpn11 is a proteasome-associated deubiquitinating enzyme that is essential for viability. Recent genetic studies showed that Rpn11 is functionally linked to Rpn10, a major multiubiquitin chain binding receptor in the proteasome. Mutations in Rpn11 and Rpn10 can reduce the level and/or stability of proteasomes, indicating that both proteins influence its structural integrity. To characterize the properties of Rpn11, we examined its interactions with other subunits in the 19S regulatory particle and detected strong binding to Rpn3. Two previously described rpn3 mutants are sensitive to protein translation inhibitors and an amino acid analog. These mutants also display a mitochondrial defect. The abundance of intact proteasomes was significantly reduced in rpn3 mutants, as revealed by strongly reduced binding between 20S catalytic with 19S regulatory particles. Proteasome interaction with the shuttle factor Rad23 was similarly reduced. Consequently, higher levels of multiUb proteins were associated with Rad23, and proteolytic substrates were stabilized. The availability of Rpn11 is important for maintaining adequate levels of intact proteasomes, as its depletion caused growth and proteolytic defects in rpn3. These studies suggest that Rpn11 is stabilized following its incorporation into proteasomes. The instability of Rpn11 and the defects of rpn3 mutants are apparently caused by a failure to recruit Rpn11 into mature proteasomes.     

Molecular shredders: how proteasomes fulfill their role

The 20S proteasome is a large, cylinder-shaped protease that is found in all domains of life and plays a crucial role in cellular protein turnover. It has multiple catalytic centers located within the hollow cavity of a molecular cage. This architecture prevents unwanted degradation of endogenous proteins and promotes processive degradation of substrates by restricting the dissociation of partially digested polypeptides. Although this kind of self-compartmentalization is generally conserved, the proteasomes of bacteria, archaea and eukaryotes show many differences in architecture, subunit composition and regulation. The structure of the 20S proteasome and its inherent role in the regulation of proteasome function are gradually being elucidated.     

Proteasomes Can Degrade a Significant Proportion of Cellular Proteins Independent of Ubiquitination

The critical role of the ubiquitin-26S proteasome system in regulation of protein homeostasis in eukaryotes is well established. In contrast, the impact of the ubiquitin-independent proteolytic activity of proteasomes is poorly understood. Through biochemical analysis of mammalian lysates, we find that the 20S proteasome, latent in peptide hydrolysis, specifically cleaves more than 20% of all cellular proteins. Thirty intrinsic proteasome substrates (IPSs) were identified and in vitro studies of their processing revealed that cleavage occurs at disordered regions, generating stable products encompassing structured domains. The mechanism of IPS recognition is remarkably well conserved in the eukaryotic kingdom, as mammalian and yeast 20S proteasomes exhibit the same target specificity. Further, 26S proteasomes specifically recognize and cleave IPSs at similar sites, independent of ubiquitination, suggesting that disordered regions likely constitute the universal structural signal for IPS proteolysis by proteasomes. Finally, we show that proteasomes contribute to physiological regulation of IPS levels in living cells and the inactivation of ubiquitin-activating enzyme E1 does not prevent IPS degradation. Collectively, these findings suggest a significant contribution of the ubiquitin-independent proteasome degradation pathway to the regulation of protein homeostasis in eukaryotes.