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.     

Selective degradation of oxidized calmodulin by the 20 S proteasome

We have investigated the mechanisms that target oxidized calmodulin for degradation by the proteasome. After methionine oxidation within calmodulin, rates of degradation by the 20 S proteasome are substantially enhanced. Mass spectrometry was used to identify the time course of the proteolytic fragments released from the proteasome. Oxidized calmodulin is initially degraded into large proteolytic fragments that are released from the proteasome and subsequently degraded into small peptides that vary in size from 6 to 12 amino acids. To investigate the molecular determinants that result in the selective degradation of oxidized calmodulin, we used circular dichroism and fluorescence spectroscopy to assess oxidant-induced structural changes. There is a linear correlation between decreases in secondary structure and the rate of degradation. Calcium binding or the repair of oxidized calmodulin by methionine sulfoxide reductase induces comparable changes in alpha-helical content and rates of degradation. In contrast, alterations in the surface hydrophobicity of oxidized calmodulin do not alter the rate of degradation by the proteasome, indicating that changes in surface hydrophobicity do not necessarily lead to enhanced proteolytic susceptibility. These results suggest that decreases in secondary structure expose proteolytically sensitive sites in oxidized calmodulin that are cleaved by the proteasome in a nonprocessive manner.     

Catalytic site-specific inhibition of the 20S proteasome by 4-hydroxynonenal

The proteasome is responsible for most intracellular protein degradation and is essential for cell survival. Previous research has shown that the proteasome can be inhibited by a number of oxidants, including 4-hydroxynonenal (HNE). The present study demonstrates that HNE rapidly inhibits the chymotrypsin-like activity of the 20S proteasome purified from liver. Subunits containing HNE-adducts were identified following 2D gel electrophoresis, Western immunoblotting, and analysis by MALDI-TOF MS. At a time when only the chymotrypsin-like activity was inhibited, the alpha 6/C2 subunit was uniquely modified. These results provide important molecular details regarding the catalytic site-specific inhibition of proteasome by HNE.     

Processing of autophagic protein LC3 by the 20S proteasome

Ubiquitin-proteasome system and autophagy are the two major mechanisms for protein degradation in eukaryotic cells. LC3, a ubiquitin-like protein, plays an essential role in autophagy through its ability to be conjugated to phosphatidylethanolamine. In this study, we discovered a novel LC3-processing activity, and biochemically purified the 20S proteasome as the responsible enzyme. Processing of LC3 by the 20S proteasome is ATP- and ubiquitin-independent, and requires both the N-terminal helices and the ubiquitin fold of LC3; and addition of the N-terminal helices of LC3 to the N terminus of ubiquitin renders ubiquitin susceptible to 20S proteasomal activity. Further, the 20S proteasome processes LC3 in a stepwise manner, it first cleaves LC3 within its ubiquitin fold and thus disrupt the conjugation function of LC3; subsequently and especially at high concentrations of the proteasome, LC3 is completely degraded. Intriguingly, proteolysis of LC3 by the 20S proteasome can be inhibited by p62, an LC3-binding protein that mediates autophagic degradation of polyubiquitin aggregates in cells. Therefore, our study implicates a potential mechanism underlying interplay between the proteasomal and autophagic pathways. This study also provides biochemical evidence suggesting relevance of the controversial ubiquitin-independent proteolytic activity of the 20S proteasome.     

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.     

Inhibition of 20 S and 26 S proteasome activity by lithium chloride: impact on the differentiation of leukemia cells by all-trans retinoic acid

Lithium affects several enzymatic activities, however, the molecular mechanisms of lithium actions are not fully understood. We previously showed that LiCl interacts synergistically with all-trans-retinoic acid to promote terminal differentiation of WEHI-3B D(+) cells, a phenomenon accompanied by the recovery of the retinoid-induced loss of retinoic acid receptor alpha protein pools. Here, we demonstrate the effects of LiCl on proteasome-dependent degradation of retinoic acid receptor alpha proteins. LiCl alone, or in combination with all-trans-retinoic acid, increased cellular levels of ubiquitinated retinoic acid receptor alpha and markedly reduced chymotryptic-like activity of WEHI-3B D(+) 20 S and 26 S proteasome enzymes. Neither KCl nor all-trans-retinoic acid affected enzyme activity, whereas NaCl produced a modest reduction at relatively high concentrations. In addition, LiCl inhibited 20 S proteasome chymotryptic-like activity from rabbits but had no effect on tryptic-like activity of the 26 S proteasome. This effect has significant consequences in stabilizing the retinoic acid receptor alpha protein levels that are necessary to promote continued differentiation of leukemia cells in response to all-trans-retinoic acid. In support of this concept, combination of proteasome inhibitors beta-clastolactacystin or benzyloxycarbonyl-Leu-Leu-Phe with all-trans-retinoic acid increased differentiation of WEHI-3B D(+) cells in a manner that was analogous to the combination of LiCl and all-trans-retinoic acid.     

Hsp90 enhances degradation of oxidized calmodulin by the 20 S proteasome

The 20 S proteasome has been suggested to play a critical role in mediating the degradation of abnormal proteins under conditions of oxidative stress and has been found in tight association with the molecular chaperone Hsp90. To elucidate the role of Hsp90 in promoting the degradation of oxidized calmodulin (CaM(ox)), we have purified red blood cell 20 S proteasomes free of Hsp90 and assessed their ability to degrade CaM(ox) in the absence or presence of Hsp90. Purified 20 S proteasome does not degrade CaM(ox) unless Hsp90 is added. CaM(ox) degradation is sensitive to both proteasome and Hsp90-specific inhibitors and is further enhanced in the presence of 2 mm ATP. Irrespective of the presence of Hsp90, we find that unoxidized CaM is not significantly degraded. Direct binding measurements demonstrate that Hsp90 selectively associates with CaM(ox); essentially no binding is observed between Hsp90 and unoxidized CaM. These results indicate that Hsp90 in association with the 20 S proteasome can selectively associate with oxidized and partially unfolded CaM to promote degradation by the proteasome.     

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.     

Characterization of the 20S proteasome from the actinomycete Frankia

Frankia is an actinomycete that fixes atmospheric nitrogen in symbiotic association with the root systems of a variety of non-leguminous plants, denominated actinorhizal plants. Information on the biology of proteolysis in Frankia is almost non-existent as it is extremely difficult to grow this organism. We have purified 20S proteasomes from Frankia strain ACN14a/ts-r. It is composed of one alpha-subunit and one beta-subunit, which assemble into the canonical structure of four rings of seven subunits each. The enzyme displayed a chymotrypsin-like activity against synthetic substrates and was sensitive to lactacystin, a specific proteasome inhibitor. Analysis of the structural genes and the flanking regions revealed a similar organization to Rhodococcus erythropolis, Mycobacterium tuberculosis and Streptomyces coelicolor and showed that the beta-subunit is encoded with a 52-amino-acid propeptide that is cleaved off in the course of the assembly. We report also for the first time the in vitro assembly of chimeric proteasomes composed of Frankia and Rhodococcus erythropolis subunits, which are correctly assembled and proteolytically active.     

Degradation of hypochlorite-damaged glucose-6-phosphate dehydrogenase by the 20S proteasome.

Glucose-6-phosphate dehydrogenase (G6PD) was treated with various concentrations of hypochlorite, which is produced by myeloperoxidase and is one of the most important oxidants during inflammatory processes. Inhibition of enzymatic activity, protein fragmentation, and proteolytic susceptibility toward the isolated 20S proteasome of G6PD were investigated. With rising hypochlorite concentrations, an increased proteasomal degradation of G6PD was measured. This occurred at higher hypochlorite concentrations than G6PD inactivation and at lower levels than G6PD fragmentation. The proteolytic activities of the 20S proteasome itself was determined by degradation of oxidized model proteins and cleavage of the synthetic proteasome substrate suc-LLVY-MCA. Proteasome activities remained intact at hypochlorite concentrations in which G6PD is maximally susceptible to proteasomal degradation. Only higher hypochlorite concentrations could decrease the proteolytic activities of the proteasome, which was accompanied by disintegration and fragmentation of the proteasome and proteasome subunits. Therefore, we conclude that the 20S proteasome can degrade proteins moderately damaged by hypochlorite and could contribute to an increased protein turnover in cells exposed to inflammatory stress.     

Age-related accumulation of non-heme ferric and ferrous iron in mouse ovarian stroma visualized by sensitive non-heme iron histochemistry

Sensitive non-heme iron histochemistry--namely, the perfusion-Perls method and perfusion-Turnbull method--was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.     

Biochemical analysis of the 20 S proteasome of Trypanosoma brucei

We describe here biochemical characterization of the 20 S proteasome from the parasitic protozoan Trypanosoma brucei. Similar to the mammalian proteasome, the T. brucei proteasome is made up of seven alpha- and seven beta-subunits. Of the seven beta-type subunits, five contain pro-sequences that are proteolytically removed during assembly, and three of them are predicted to be catalytic based on primary sequence. Affinity labeling studies revealed that, unlike the mammalian proteasome where three beta-subunits were labeled by the affinity reagents, only two beta-subunits of the T. brucei proteasome were labeled in the complex. These two subunits corresponded to beta2 and beta5 subunits responsible for the trypsin-like and chymotrypsin-like proteolytic activities, respectively. Screening of a library of 137,180 tetrapeptide fluorogenic substrates against the T. brucei 20 S proteasome confirmed the nominal beta1-subunit (caspase-like or PGPH) activity and identified an overall substrate preference for hydrophobic residues at the P1 to P4 positions in a substrate. This overall stringency is relaxed in the 11 S regulator (PA26)-20 S proteasome complex, which shows both appreciable activities for cleavage after acidic amino acids and a broadened activity for cleavage after basic amino acids. The 20 S proteasome from T. brucei also shows appreciable activity for cleavage after P1-Gln that is minimally observed in the human counterpart. These results demonstrate the importance of substrate sequence specificity of the T. brucei proteasome and highlight its biochemical divergence from the human enzyme.     

Dissecting beta-ring assembly pathway of the mammalian 20S proteasome

The 20S proteasome is the catalytic core of the 26S proteasome. It comprises four stacked rings of seven subunits each, alpha(1-7)beta(1-7)beta(1-7)alpha(1-7). Recent studies indicated that proteasome-specific chaperones and beta-subunit appendages assist in the formation of alpha-rings and dimerization of half-proteasomes, but the process involved in the assembly of beta-rings is poorly understood. Here, we clarify the mechanism of beta-ring formation on alpha-rings by characterizing assembly intermediates accumulated in cells depleted of each beta-subunit. Starting from beta2, incorporation of beta-subunits occurs in an orderly manner dependent on the propeptides of beta2 and beta5, and the C-terminal tail of beta2. Unexpectedly, hUmp1, a chaperone functioning at the final assembly step, is incorporated as early as beta2 and is required for the structural integrity of early assembly intermediates. We propose a model in which beta-ring formation is assisted by both intramolecular and extrinsic chaperones, whose roles are partially different between yeast and mammals.     

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.     

Selective degradation of ubiquitinated Sic1 by purified 26S proteasome yields active S phase cyclin-Cdk.

Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 activated S-Cdk only partially in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within approximately 2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was approximately 200-fold slower. We propose that the 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.     

Visualization of non-heme ferric and ferrous iron by highly sensitive non-heme iron histochemistry in the stress-induced acute gastric lesions in the rat

Redox-active non-heme iron catalyzes hydroxyl radical [Formula: see text] generation through Haber-Weiss reaction. Oxidative tissue damage by OH* has been suggested in the development of stress-induced gastric lesion. Using highly sensitive non-heme iron histochemistry, the perfusion-Perls and -Turnbull methods plus DAB intensification, we studied the distribution of non-heme ferric and ferrous iron (NHF[III] and NHF[II]) in the normal stomach and its changes in the acute gastric lesions induced by restraint water immersion (RWI) stress in the rat. Both NHF[III] and NHF[II] staining increased in the oncotic parietal cells located at the erosive lesion which developed on the gastric mucosal folds after 3 h RWI. It was considered that increase in non-heme iron in these cells catalyzed OH* generation under the presence of O(2)(*-) released from abundant injured mitochondria. This was supported by the increase in H(2)O(2) staining in the erosive region and the obvious reduction of the gastric lesion following administration of deferoxamine before RWI. NHF[II] was stained in the arterial endothelium in the tela submucosa of the normal gastric wall and increase in the entire gastric mucosa after 3 h RWI suggests that the changes in the vascular non-heme iron metabolism were also involved in the response of the stomach to stressful conditions.