Purification of the eukaryotic 20S proteasome.

The 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell. Combination of the 20S proteasome with a complex of regulatory proteins forms the 26S proteasome, which in turn is responsible for the recognition and degradation of ubiquitin-protein conjugates. As described in this unit, the constitutive form of the 20S proteasome can be conveniently purified as a stable and homogeneous preparation from bovine pituitaries. A support protocol details an enzyme assay used in evaluating proteasomal activity. The 20S proteasome is the catalytic core of the major extralysosomal proteolytic system of the cell.

Properties of the beta subunit of the proteasome activator PA28 (11S REG).

The proteasome activator PA28 (11S REG) is composed of two homologous subunits termed alpha and beta. The properties of the recombinant beta-subunit were explored and compared to the properties of the recombinant alpha-subunit. PA28beta produced in an Escherichia coli expression system migrates on a calibrated gel filtration column as an apparent heptamer (Mr = 250,000). Low concentrations of SDS (0.005%), dissociate the protein to a monomer (Mr = 33,000). PA28beta, has a complex effect on proteasome activity. At concentrations which favor oligomerization (> 2 microM), PA28beta is a strong proteasome activator although its affinity for the proteasome is about 10-fold less than recombinant PA28alpha. The catalytic properties of the PA28alpha and PA28beta-activated proteasome are similar. At low concentrations, PA28beta is a monomer and a potent allosteric proteasome inhibitor. These studies show that oligomerization of PA28beta is required for proteasome activation and that PA28beta monomers are potent proteasome inhibitors.

Properties of the nuclear proteasome activator PA28gamma (REGgamma).

PA28 or 11S REG is a proteasome activator composed of homologous alpha- and beta-subunits and predominantly found in the cytosol. A homologous protein originally known as the Ki antigen but now called PA28gamma or REGgamma is predominantly localized in the nucleus. To further characterize the biochemical properties of PA28gamma, we expressed and purified homogenous recombinant human protein with and without an N-terminal 6-His extension. PA28gamma is a heptamer based on the molecular masses of the native and monomeric proteins. The heptameric 6-His fusion protein can dimerize. Recombinant PA28y stimulates the proteasome-mediated hydrolysis of synthetic substrates containing hydrophobic, basic, and acidic amino acids in the P1 position. Stimulation is dependent on substrate size. PA28y only minimally stimulates degradation of the oxidized B chain of insulin. PA28gamma may facilitate the later stages of protein metabolism in the nucleus and/or have a more specialized role in controlling the levels of biologically active peptides in the nucleus.

Modulators of the activation of the proteasome by PA28 (11S reg).

The degradation of chromogenic substrates and oligopeptides by the 20S proteasome is markedly enhanced and the generation of antigens for presentation by the MHC class-I system is facilitated by combination with an activator protein known as PA28 or 11S reg. We have described the properties of a PA28-proteasome modulator, N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol which shifts the pathway of peptide hydrolysis by the activated proteasome to products terminating in an acidic amino acid at the expense of products terminating in a hydrophobic amino acid. We now report that piperazinyl phenothiazines and several other antipsychotic drugs modulate the PA28-20S activated proteasome in an opposite manner. Fluphenazine, trifluoperazine and prochlorperazine antagonize the peptidylglutamyl peptide bond hydrolyzing activity of the activated proteasome much more strongly than the chymotrypsinlike activity. The chicken ovalbumin immunodominant epitope SIINFEKL is degraded by the activated proteasome to SIINFE and SIINF in approximately equimolar amounts. Piperazinyl phenothiazines promote formation of SIINF whereas Psi-ol promotes formation of SIINFE. PA28- proteasome modulators by modifying the profile of peptides produced by the activated proteasome, may either enhance or suppress the immune response.

Modulation of the PA28alpha-20S proteasome interaction by a peptidyl alcohol.

The peptidyl alcohol N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinol is a mild activator of the chymotrypsin-like activity of the proteasome. When added to an incubation mixture of recombinant PA28alpha plus 20S proteasome the peptidyl alcohol antagonizes the stimulation of the chymotrypsin-like activity by PA28alpha in a dose-dependent manner (IC50 = 30 microM). This effect is selective for the chymotrypsin-like activity. Stimulation of the peptidyl-glutamyl peptide bond hydrolyzing activity of the proteasome by PA28alpha is not affected by the peptidyl alcohol. The ovalbumin immunodominant epitope SIINFEKL is hydrolyzed by the PA28alpha-activated 20S proteasome to SIINF and SIINFE in approximately equimolar amounts. Addition of the peptidyl alcohol to an incubation mixture of PA28alpha, 20S proteasome and SIINFEKL shifts the ratio of products in favor of SIINFE. A similar shift in favor of postglutamyl cleavages occurs with the extended peptide LEQLESIINFEKLTE. By altering the ratio of products produced by the PA28alpha-activated proteasome, the peptidyl alcohol acts as a proteasome modulator. Proteasome modulators represent a novel class of molecules with a potential for altering the processing of antigens by the PA28-proteasome complex for presentation by the MHC class I system.

Properties of the proteasome activator subunit PA28 alpha and its des-tyrosyl analog.

The proteasome activator protein PA28 or 11 S regulator may play an important role in facilitating the generation of peptides for presentation by the MHC class I system. PA28 is composed of two homologous subunits termed alpha and beta. Removal of the carboxyl terminal tyrosine of the alpha subunit of PA28 abolishes activity (X. Song et al., 1997, J. Biol. Chem. 272, 27994-28000). To explore the structural basis of this effect the des-tyrosyl analog of PA28alpha prepared by site-directed mutagenesis and PA28alpha were expressed at high levels in a baculovirus system and purified by FPLC. Des-tyrosyl-PA28alpha neither stimulated the proteasome nor competed with PA28alpha for binding to the proteasome. Hydrophobic interaction chromatography revealed that the hydrophobicity of the mutant protein was considerably greater than PA28alpha. When the mutant protein was chromatographed on a calibrated Superose 6 column a mixture of approximately 25% oligomer and 75% monomer was found. The oligomer weakly stimulated the proteasome but this molecule was labile. Very low concentrations of SDS (0.005%) dissociated PA28alpha and abolished its stimulatory activity. It is concluded that the lack of activity of des-tyrosyl-PA28alpha is due to conformational changes resulting in dissociation and that the oligomeric form of PA28alpha is required for activation.

Synthetic peptide-based activators of the proteasome.

The development of small molecule peptide-based activators of the 20S proteasome or multicatalytic proteinase complex was initiated. The enhancement of antigen presentation by transfection of the protein activator PA28alpha into a mouse fibroblast cell line [10] supports the potential use of small molecule activators in stimulating the immune response. Four classes of peptide-based activators were synthesized, i.e. peptidyl alcohols, esters, p-nitroanilides and nitriles. These compounds markedly and reversibly stimulated the hydrolysis of suc-LLVY-MCA, Z-LLE-NA and Z-GPALG-p-aminobenzoate as well as hydrolysis of the decapeptide angiotensin I. Stimulation was due to a decrease in the Km and increase in the Vmax of the substrate. In general, the EC50 for activation ranged from 50-150 mM and maximal stimulation varied from 3 to 15 fold depending on the activity measured. Z-IE(Ot-Bu)AL-p-nitroanilide, a proteasome substrate, markedly stimulated the hydrolysis of Z-GPALG-pAB by binding to a saturable high affinity site distinct from its binding site as substrate. Since all effective activators contain hydrophobic groups in positions P1-P5, low aqueous solubility is a limitation of these compounds. Competition experiments suggest that these activators bind to the same site as PA28.

Cleavage of Pro-X and Glu-X bonds catalyzed by the branched chain amino acid preferring activity of the bovine pituitary multicatalytic proteinase complex (20S proteasome).

The multicatalytic proteinase complex or 20S proteasome is involved in the extralysosomal degradation of both long- and short-lived proteins. The eukaryotic enzyme is composed of 14 nonidentical subunits arranged as a complex dimer of the composition (alpha7beta7)2. Recent studies identify N-terminal threonines present on some beta-subunits as the active-site residues. It has been proposed that the molecule contains three or four proteolytically active subunits [Seemuller et al., Science 268, 579-582 (1995)]. Studies with synthetic substrates, activators, and inhibitors, however, have identified at least five distinct catalytic activities. To further characterize the specificity of the previously defined "peptidyl glutamyl peptide bond hydrolyzing activity," N-benzyloxycarbonyl-Leucyl-Leucyl-Glutamal was synthesized as a potential inhibitor. Surprisingly, this aldehyde most potently inhibited the "branched chain amino acid preferring activity" (BrAAP). To further explore BrAAP specificity, novel substrates containing internal prolyl and glutamyl residues were synthesized. Their use established that the BrAAP activity catalyzed both a postproline and a postglutamate cleavage and therefore has a broader specificity than previously recognized. These results help explain earlier observations on treatment of the multicatalytic proteinase complex with 3,4-dichloroisocoumarin. This reagent activates both the BrAAP activity and the degradation of beta-casein and inhibits the other catalytic activities.

The antitumor drug aclacinomycin A, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsin-like activity of the bovine pituitary 20 S proteasome.

The antitumor drug aclacinomycin A was previously shown to inhibit the degradation of ubiquitinated proteins in rabbit reticulocyte lysates with an IC50 of 52 microM (Isoe, T., Naito, M., Shirai, A., Hirai, R., and Tsuruo, T.(1992) Biochim. Biophys. Acta 1117, 131-135). We report here that from all the catalytic activities of the 20 S proteasome tested, the chymotrypsin-like activity was the only one affected by the antitumor drug. An important requirement for inhibition of the chymotrypsin-like activity seemed to be the presence of hydrophobic nonpolar residues in positions P1 to P3. Degradation of Z-E(OtBu)AL-pNA and Z-LLL-AMC at pH 7.5 was dramatically (87-98%) inhibited by 50 microM of the drug, while that of Z-GGL-pNA (containing uncharged polar residues in positions P2 and P3) and succinyl-LLVY-AMC (containing an uncharged polar residue in the P1 position) was inhibited only 11 and 24%, respectively. Aclacinomycin A had no effect on cathepsin B, stimulated trypsin, and inhibited chymotrypsin and, to a lesser extent, calpain. The aglycone and sugar moieties of the cytotoxic drug are essential for inhibition. The results presented here support a major role for the chymotrypsin-like activity in the degradation of ubiquitinated proteins. Aclacinomycin A is the first described non-peptidic inhibitor showing discrete selectivity for the chymotrypsin-like activity of the 20 S proteasome.

A novel chymotrypsin-like component of the multicatalytic proteinase complex optimally active at acidic pH.

The multicatalytic proteinase complex (MPC) or proteasome is a multimeric, high-molecular-weight (700,000), extralysosomal proteolytic enzyme found in eukaryotes and in archaebacteria. Its multiple catalytic sites grant it a broad cleavage specificity toward short peptides and protein substrates. The pH optima of the catalytic activities of MPC are in the neutral or slightly alkaline range. We present here evidence for cryptic catalytic components of MPC optimally active at an acidic pH. Studies with a hydrophobic fluorescent probe provide direct evidence for conformational changes brought about by exposing the complex to an acidic environment. One of the newly described components, designated "acidic chymotrypsin-like activity," cleaves the Leu-2-naphthylamide bond in the substrate Boc-Val-Glu-Ala-Leu-2-naphythylamide. Compared with the classical "neutral" chymotrypsin-like activity defined by cleavage of the Leu-p-nitroanilide bond in Z-Gly-Gly-Leu-p-nitroanilide, the newly described component is not inhibited by monovalent cations and is less sensitive to the peptidyl aldehyde Z-Gly-Gly-leucinal, an inhibitor of the neutral chymotrypsin-like activity. In addition, we describe the properties of a novel potent peptidyl aldehyde, Z-Ile-Glu(OtBu)-Ala-leucinal, which is an inhibitor of both the acidic and neutral chymotrypsin-like activities of MPC, with IC50 values of 0.25 and 6.5 microM, respectively. In the presence of 65 microM of the newly synthesized peptidyl aldehyde, other MPC components such as the trypsin-like and peptidyl-glutamyl peptide hydrolyzing activities were decreased only by 14 and 9%, respectively. The hydrophobicity, potency, and specificity of Z-Ile-Glu(OtBu)-Ala-leucinal toward the chymotrypsin-like activities of the complex make it a valuable pharmacological tool with which to investigate the physiological roles of MPC.

Immune responses in newly developed short-lived SAM mice. III. Genetic control of defective helper T-cell activity in in vitro primary antibody response.

Immune activities of newly developed, short-lived SAM-P/1 mice declined sharply after a few months of age. As early as 2 months of age, the activity of T-helper (Th) cells ('Th2'-like) in the in vitro primary antibody response was profoundly impaired, in contrast to normal activity of Th cells ('Th1'-like) engaged in cell-mediated immune responses. Thus, young SAM-P/1 mice show a functional heterogeneity of Th cells. To determine how such a 'Th2' abnormality is inherited in SAM-P/1 mice, immune activities of their hybrids and backcrosses between MHC-identical, high responder B10.BR mice were statistically assessed. The distribution of responses did not support the Mendelian single-gene determination for low responsiveness. Moreover, involvement of a single gene which exhibits incomplete dominance was ruled out because of a continuous distribution pattern of antibody response in the F2 generation. Such an analysis strongly suggests that the impaired 'Th2'-like activity of SAM-P/1 mice is under control of two genes, based on the proportion of low responders in F2 hybrids (29 out of 267, 10.8%) and on calculation according to Wright's formula (n = 1.72). Further linkage analyses suggest that one of the genes is closely linked to albino coat-colour (c) locus on chromosome 7. The putative two genes are likely to control 'differentiation' or 'maturation' of Th2-like cells defectively, but the defect is not refractory, because in vivo-primed Th cells function in vitro as do those in ordinary strains of mice. Possible mechanisms and biological significance in relation to loss of immune activity with ageing are discussed.