The proteasome inhibitor PI31 competes with PA28 for binding to 20S proteasomes.

PI31 is a previously described inhibitor of 20S proteasomes. Using recombinant PI31 we have analyzed its effect on proteasomal hydrolyzing activity of short fluorogenic substrates and of a synthetic 40-mer polypeptide. In addition, we investigated its influence on the activation of 20S proteasome by the proteasome activator PA28. PI31 inhibits polypeptide degradation already at concentrations which only partially inhibit fluorogenic substrate turnover and immunosubunits do not influence the PI31 binding affinity. Furthermore our data demonstrate that PI31 is a potent competitor of PA28-mediated activation.     

20S proteasome, hsp90, p97 fusion protein, PA28 activator copurifying oligomers and ATPase activities

Whether hsp90 acts in an ATP-dependent or independent way is of crucial importance for understanding the molecular mechanism of this chaperone and, to day, the involvement of ATP hydrolysis in hsp90 function is still a controversial subject. ATPase activities may be detected in partially purified hsp90's preparations from rabbit muscle. We demonstrate that the major contaminant associated with hsp90 is the p97 fusion protein and that these oligomeric structures are copurifying together with the 20S proteasome and its PA28 activator. Improving the purification procedure permits to separate hsp90 and p97 to homogeneity. Then, our attempts failed to detect any significant ATPase activity in the hsp90 fraction. Thus, p97 would be principally responsible for the ATPase activity detected in partially purified hsp90 preparations from rabbit muscle.     

Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes

PA28 is a gamma-interferon-induced complex that associates with the 20S proteasome and stimulates breakdown of small peptides. Recent immunoprecipitation studies indicate that, in vivo, PA28 also exists in larger complexes that also contain the 19S particle, which is required for ATP-ubiquitin-dependent degradation of proteins. However, because of its lability, the structure and properties of this larger complex remain unclear. Here, we demonstrate that, in vitro, PA28 can associate with 'singly capped' 26S (i.e. 19S-20S) proteasomes. Electron microscopy of the resulting structures revealed one PA28 ring at one end of the 20S particle and a 19S complex at the other. These hybrid complexes show enhanced hydrolysis of small peptides, but no significant increase in rates of protein breakdown. Nevertheless, during breakdown of proteins, the complexes containing PA28alphabeta or PA28alpha generated a pattern of peptides different from those generated by 26S proteasomes, without altering mean product length. Presumably, this change in peptides produced accounts for the capacity of PA28 to enhance antigen presentation.     

The C-terminal extension of the beta7 subunit and activator complexes stabilize nascent 20 S proteasomes and promote their maturation

The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. Beta7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4 C terminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.     

Proteasome activator subunit PA28 alpha and related Ki antigen (PA28 gamma) are absent from the nuclear fraction purified by sucrose gradient centrifugation

The aim of the present work was to attempt to partially purify PA28 (REG) alpha and gamma (Ki antigen) in the nuclear fraction from NT2/D1 cells. Nuclei were isolated by the hypertonic sucrose gradient centrifugation method and fractionated into membrane/nucleoplasmic and chromatin/nucleolar fractions. Western blotting with anti-histone and anti-beta-tubulin monoclonal antibodies confirmed the accuracy of the procedure. Proteasomes were present mainly in the cytoplasm but also in the nuclei. Disruption of the nuclear envelope released the proteasomes implying a loose or no binding with the chromatin. PA28 alpha and gamma were detected mainly in the cytosol and to a lesser extent in the crude nuclear pellet, however the purified nuclei were devoid of PA28 alpha and gamma. This indicates, that only a small fraction of the PA28 activator is present in the nuclei as detected by immunofluorescence or/and it is easily removed during nuclear purification.     

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

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

Effect of quercetin on the activity of purified 20S and 26S proteasomes and proteasomal activity in isolated cardiomyocytes

Quercetin inhibits in vitro in dose-dependent manner all three peptidase activities in purified 20S proteasome, the inhibitory effect is comparable to that of a specific proteasome inhibitor. The maximum inhibitory effect of quercetin was observed against the chymotrypsin-like activity of 20S proteasome. Similarly, quercetin inhibits the activity of 26S proteasome from proteasomal fraction II (PF II). Determination of proteasome activity in isolated cardiomyocytes has demonstrated 26% inhibition of trypsin-like proteasomal activity (p = 0.03), 63.7% inhibition of chymotrypsin-like activity (p = 0.04), and 34.2% inhibition of peptidyl-glutamyl peptide hydrolase (p = 0.16) activity by quercetin. Quercetin, its water-soluble analogue corvitin, and clastolactacystin-β-lactone, the specific proteasome inhibitor, exert virtually the same effects on cardiomyocytes. At the concentrations of 5 and 10 μM quercetin corvitin caused the decrease in number of living cardiomyocytes and the increase in number of necrotic and apoptotic cells. At the concentration of 2.5 μM quercetin and corvitin reduced substantially the damaging effect of anoxia-reoxygenation on cardiomyocytes and resulted in decrease in number of necrotic and apoptotic cells. The data obtained suggest that mechanisms of the quercetin cardioprotective effect may involve the inhibition of proteasome activity.     

Reconstitution of Bacillus stearothermophilus 50 S ribosomal subunits from purified molecular components.

Bacillus stearothermophilus 50 S ribosomal subunits have been reconstituted from a mixture of purified RNA and protein components. The protein fraction of 50 S subunits was separated into 27 components by a combination of various methods including ion exchange and gel filtration chromatography. The individual proteins showed single bands in a variety of polyacrylamide gel electrophoresis systems, and nearly all showed single spots on two-dimensional polyacrylamide gels. The molecular weights of the proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An equimolar mixture of the purified proteins was combined with 23 S RNA and 5 S RNA to reconstitute active 50 S subunits by the procedure of Nomura and Erdmann (Nomura, M., and Erdmann, V. A. (1970) Nature 226, 1214-1218). Reconstituted 52 S subunits containing purified proteins were slightly more active than subunits reconstituted with an unfractionated total protein extract in poly(U)-dependent polyphenylalanine synthesis and showed comparable activity in various assays for ribosomal function. The reconstitution proceeded more rapidly with the mixture of purified proteins than with the total protein extract. Reconstituted 50 S subunits containing purified proteins co-sedimented with native 50 S subunits on sucrose gradients and had a similar protein compsoition. Initial experiments on the roles of the individual proteins in ribosomal structure and function were performed. B. stearothermophilus protein 13 was extracted from 50 S subunits under the same conditions as escherichia coli L7/L12, and the extraction had a similar effect on ribosomal function. When single proteins were omitted from reconstitution mixtures, in most cases the reconstituted 50 S subunits showed decreased activity in polypheylalanine synthesis.     

Identification, purification, and characterization of a protein activator (PA28) of the 20 S proteasome (macropain).

A protein that greatly stimulates the multiple peptidase activities of the 20 S proteasome (also known as macropain, the multicatalytic protease complex, and 20 S protease) has been purified from bovine red blood cells and from bovine heart. The activator protein was a single polypeptide with an apparent molecular weight of 28,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and had a native molecular weight of approximately 180,000. This protein, which we have termed PA28, regulated all three of the putatively distinct peptidase activities displayed by each of two functionally different forms of the proteasome. This regulation usually included both an increase in the maximal reaction velocity and a decrease in the concentration of substrate required for half-maximal velocity and indicated that PA28 acted as a positive allosteric effector of the proteasome. PA28 failed, however, to stimulate the hydrolysis of large protein substrates such as casein and lysozyme. These results suggested that the hydrolysis of protein substrates occurred at a site or sites distinct from those that hydrolyzed small peptides and that the regulation of the two processes could be uncoupled. Evidence for direct binding of PA28 to the proteasome was obtained by glycerol density gradient centrifugation. PA28 may play an important regulatory role in intracellular proteolytic pathways mediated by the proteasome.     

Reconstitution of the activities of the RecBCD holoenzyme of Escherichia coli from the purified subunits.

The Escherichia coli RecBCD holoenzyme and the individual constituent subunits have been purified from overproducing strains. The purified RecBCD holoenzyme has a native molecular mass of approximately 330 kDa, indicative of a heterotrimer subunit assembly. The RecB, RecC, and RecD subunits can associate in vitro to give nuclease, helicase, ATPase, and Chi-specific endonuclease activities which are indistinguishable from those of the RecBCD holoenzyme. At concentrations at which the reconstituted RecB + C + D enzyme is very active, none of the individual RecB, RecC, or RecD subunits have readily detectable activities of the holoenzyme, except RecB protein which had previously been shown to exhibit DNA-dependent ATPase activity (Hickson, I. D., Robson, C. N., Atkinson, K. E., Hutton, L., and Emmerson, P. T. (1985) J. Biol. Chem. 260, 1224-1229). At higher concentrations and with shorter DNA substrates reconstituted RecBC protein exhibits low levels of helicase and exonuclease activity.     

The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions

Proteasomes are cylindrical structures that function in multiple cellular processes by degrading a wide variety of cytosolic and nuclear proteins. Substrate access and product release from the enclosed catalytic chamber occurs through axial pores that are opened by activator complexes. Here, we report high-resolution structures of wild-type and mutant archaeal proteasomes bound to the activator PA26. These structures support the proposal that an ordered open conformation is required for proteolysis and that its formation can be triggered by outward displacement of surrounding residues. The structures and associated biochemical assays reveal the mechanism of binding, which involves an interaction between the PA26 C terminus and a conserved lysine. Surprisingly, biochemical observations implicate an equivalent interaction for the unrelated ATP-dependent activators PAN and PA700.     

Nitric oxide decreases activity and levels of the 11S proteasome activator PA28 in the vasculature

The 11S proteasome activator (PA28) binds to the 20S proteasome and increases its ability to degrade small peptides. Expression of PA28 subunits (α, β, γ) is induced by interferon-γ stimulation. Inflammation plays a role in the development of neointimal hyperplasia, and we have previously shown that nitric oxide (NO) reduces neointimal hyperplasia in animal models and 26S proteasome activity in rat aortic smooth muscle cells (RASMC). Here, we show that PA28 increased 26S proteasome activity in RASMC, as measured by a fluorogenic assay, and the NO donor S-nitroso N-acetylpenicillamine significantly inhibits this activation. This effect was abrogated by the reducing agents dithiothreitol and HgCl(2), suggesting that NO affects the activity of PA28 through S-nitrosylation. NO did not appear to affect PA28 levels or intracellular localization in RASMC in vitro. Three days following rat carotid artery balloon injury, levels of PA28α, β and γ subunits were decreased compared to uninjured control arteries (n=3/group) in vivo. The NO donor proline NONOate further decreased PA28α, β and γ levels by 1.9-, 2.3- and 3.4-fold, respectively, compared to uninjured control arteries. Fourteen days following arterial injury, levels of PA28α, β and γ subunits were increased throughout the arterial wall compared to uninjured control arteries, but were greatest for the α and β subunits. NO continued to decrease the levels of all three PA28 subunits throughout the arterial wall at this time point. Since the PA28 subunits are involved in the breakdown of peptides during inflammation, PA28 inhibition may be one mechanism by which NO inhibits neointimal hyperplasia.     

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.     

Expression of subunits of the 19S complex and of the PA28 activator in rat skeletal muscle

A precise knowledge of the role of subunits of the 19S complex and the PA28 regulator, which associate with the 20S proteasome and regulate its peptidase activities, may contribute to design new therapeutic approaches for preventing muscle wasting in human diseases. The proteasome is mainly responsible for the muscle wasting of tumor-bearing and unweighted rats. The expression of some ATPase (MSS1, P45) and non ATPase (P112-L, P31) subunits of the 19S complex, and of the two subunits of the PA28 regulator, was studied in such atrophying muscles. The mRNA levels for all studied subunits increased in unweighted rats, and analysis of MSS1 mRNA distribution profile in polyribosomes showed that this subunit entered active translation. By contrast, only the mRNA levels for MSS1 increased in the muscles from cancer rats. Thus, gene expression of the proteasome regulatory subunits depends on a given catabolic state. Torbafylline, a xanthine derivative which inhibits tumor necrosis factor production, prevented the activation of protein breakdown and the increased expression of 20S proteasome subunits in cancer rats, without reducing the elevated MSS1 mRNA levels. Thus, the increased expression of MSS1 is regulated independently of 20S proteasome subunits, and did not result in accelerated proteolysis.     

Global organization and function of mammalian cytosolic proteasome pools: Implications for PA28 and 19S regulatory complexes

Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S alpha-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle.     

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 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.     

Toxoplasma gondii: ultrastructure and antigenicity of purified tachyzoite pellicle.

When purified Toxoplasma gondii tachyzoites were treated with hemolysin, DNase, and RNase, the organisms yielded a three-component system containing the outer membrane (pellicle), microtubules, and conoid in relatively normal morphological configuration. Further treatment of this preparation with protease digested all but the pellicle which was more collapsed in appearance. These two preparations were used in rabbit anti-toxoplasma and goat anti-rabbit ferritin labeling experiments. The three-component system showed ferritin label on the conoid and equal ferritin label on the outer and inner surfaces of the pellicle. The microtubules were unlabeled. The pellicle after protease treatment was labeled equally on its outer and inner surfaces, which indicated that the rabbit anti-toxoplasma serum contained antibodies against antigens on the outer and inner surfaces of the pellicle.     

Studies on histone oligomers. V. Reconstitution of chromatin from purified DNA and acid-extracted histones

DNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted. No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful. This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea. If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.