Search

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 potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of beta-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC50 values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.

The eukaryotic multicatalytic proteinase complex (proteasome) is a high molecular mass enzyme which contains 13-15 nonidentical subunits of similar size (molecular masses of 21-31 kDa), but differing widely in net charge (isoelectric points ranging from 3 to 10). At least four catalytic components termed chymotrypsin-like, trypsin-like, peptidylglutamyl peptide-hydrolyzing, and caseinolytic are associated with the proteinase. The catalytic nature of the components is unknown, since sequences of cloned subunits bear no homology to known proteinases and proteolytically active subunits have not been isolated. Analysis of the relationship between structure and catalytic function would be greatly facilitated if a means for reversibly dissociating and reassociating the proteinase were available. We provide the first evidence of reassembly of dissociated multicatalytic proteinase complex into a functional molecule. Incubation with the organic mercurial, p-chloromercuribenzoic acid disrupts in a concentration-dependent manner the quaternary structure of the enzyme, leading to formation of a heterogeneous population of subunits. Dissociation of the complex coincides with progressive loss of chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. The caseinolytic activity of the residual undissociated enzyme is markedly activated. Exposure of the dissociated enzyme to dithiothreitol restores the catalytic profile and reassociates the enzyme. Evidence for catalytically active subcomplexes was not obtained indicating that structural integrity may be necessary for expression of all defined activities.

The multicatalytic proteinase complex (proteasome) contains at least four distinct active sites catalyzing the degradation of selected chromogenic substrates (trypsin-like, chymotrypsin-like, and peptidylglutamyl peptide hydrolyzing activities) and proteins such as beta-casein. Oxidized insulin B chain was recently proposed as a model substrate for protein degradation by the multicatalytic proteinase complex (Dick, L. R., Moomaw, C. R., DeMartino, G. N., and Slaughter, C. A. (1991) Biochemistry 30, 2725-2734). We studied the dialysis-induced activation of the hydrolysis of oxidized insulin B chain by this enzyme. Removal of EDTA from purified preparations of bovine pituitary multicatalytic proteinase complex by dialysis against Tris-HCl buffers led to marked changes in the catalytic properties and structure of the enzyme. Dialysis produced a time-dependent activation of oxidized insulin B chain hydrolysis with predominant cleavage at the Glu13-Ala14 bond. A new chromogenic assay was developed for measurement of this activity. Activation was accompanied by a virtually total inactivation of the chymotrypsin-like, trypsin-like, and peptidylglutamyl peptide hydrolyzing activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a loss of the 24-kDa subunit and the appearance of a new band at 21 kDa. Amino-terminal amino acid analysis established that the 21-kDa band was autolytically derived from the 24-kDa subunit. Evidence for partial dissociation and/or aggregation indicated that autolysis destabilizes the complex. By altering the profile of catalytic activities of the multicatalytic proteinase complex, autolysis may serve as a mechanism for regulation of this macromolecule.

The breakdown of beta-casein (caseinolytic activity) by the bovine pituitary multicatalytic proteinase complex (MPC) is initiated by a fourth active site different from the previously described chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide, where Cbz is benzyloxycarbonyl), trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide), and peptidylglutamyl peptide bond-hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) (Yu, B., Pereira, M. E., and Wilk, S. (1991) J. Biol. Chem. 266, 17396-17400). 3,4-Dichloroisocoumarin, a serine proteinase inhibitor, stimulated the caseinolytic activity of bovine pituitary or lens MPC, 3-18-fold under conditions under which the other three catalytic activities were inactivated. Addition of hydroxylamine to the modified enzyme did not reverse the effects of the inhibitor. A form of the proteinase exhibiting only 2-4% of control chymotrypsin-like, trypsin-like, and PGP activities degraded beta-casein with no accumulation of intermediate peptides. 3,4-Dichloroisocoumarin, by reacting with the chymotrypsin-like, trypsin-like, and/or PGP-active sites, may promote a conformational change of MPC, rendering the caseinolytic active site accessible to the substrate. Once bound to the active site, beta-casein is rapidly degraded either by the caseinolytic component itself or by a cooperative interaction with catalytic centers that are not affected by the serine proteinase inhibitor. These results imply that the caseinolytic component does not belong to the class of serine proteinases. Other proteins tested were not degraded by the 3,4-dichloroisocoumarin-treated enzyme, suggesting that the conformation of beta-casein may be more adequate for degradation by the caseinolytic component.

The effect of magnesium ions on the catalytic activities of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. Mg2+ markedly stimulated the breakdown of dephosphorylated beta-casein (caseinolytic activity) and the hydrolysis of Cbz-Leu-Leu-Glu-2-naphthylamide (peptidylglutamyl peptide bond hydrolyzing activity) by a 1700-fold purified preparation of MPC. Cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide (trypsin-like activity) was strongly inhibited and cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide (chymotrypsin-like activity) was weakly inhibited. Similar results were produced when enzymatic activities in the absence of Mg2+ were measured at 52 degrees C rather than at 37 degrees C. Trace protein impurities were removed by phenyl-Sepharose chromatography. This additional chromatographic step, while not changing the specific activities of hydrolysis of the three synthetic chromogenic substrates, led to a marked activation of the breakdown of dephosphorylated beta-casein. Mg2+ was not able to further stimulate the caseinolytic activities of either the phenyl-Sepharose-treated preparation or the preparation measured at 52 degrees C. Mg2+ therefore converts a "repressed" form of MPC to an "activated" form, possibly by promoting dissociation of a protein inhibitor, and may serve as a physiological regulator of this enzyme complex.

The effect of N-acetylimidazole, a mild acetylating reagent, on the catalytic activities and subunit structure of the bovine pituitary multicatalytic proteinase complex (MPC) was studied. The trypsin-like activity (cleavage of Cbz-D-Ala-Leu-Arg-2-naphthylamide) and the peptidylglutamyl-peptide bond hydrolyzing (PGP) activity (cleavage of Cbz-Leu-Leu-Glu-2-naphthylamide) of MPC were rapidly inactivated by N-acetylimidazole, whereas the chymotrypsin-like activity (cleavage of Cbz-Gly-Gly-Leu-p-nitroanilide) was inactivated slowly. However, the hydrolysis of casein was markedly stimulated. Hydrolysis of casein by the acetylated enzyme generated a stable intermediate (21 kDa) which could be further degraded by native MPC. Treatment of acetylated MPC with hydroxylamine reversed the changes in trypsin-like and caseinolytic activities but did not restore the PGP activity. N-Acetylimidazole did not dissociate MPC but altered its migration on nondissociating gels presumably by acetylation of epsilon-amino groups of lysine residues. Hydroxylamine did not alter the gel electrophoretic appearance of the acetylated enzyme. These results indicate that acetylation of thiol or tyrosyl groups changes the trypsin-like and caseinolytic activities, and that amino group acetylation inhibits the PGP activity. Degradation of casein by MPC appears to be a sequential process with initial cleavage catalyzed by a component distinct from the chymotrypsin-like, trypsin-like, and PGP activities. The latter three components likely participate in the secondary proteolysis of the generated intermediates.

The multicatalytic proteinase complex (MPC) constitutes a major nonlysosomal proteolytic system that may play an important role in the processing of biologically active peptides and enzymes, as well as in intracellular metabolism. We report that at least two of its subunits of MW 28,800 (S2) and 27,000 (S3) are phosphorylated by a cAMP-dependent protein kinase (PK-A) that copurifies with the complex isolated from bovine pituitaries. The cAMP-induced phosphorylation was time dependent and inhibited by a PK-A inhibitor. Although not an integral part of the complex, PK-A activity was still present even in 1700-fold-purified and apparently homogeneous preparations by criteria of nondissociating polyacrylamide gel electrophoresis. Furthermore, we present evidence that the copurification of the two enzymes is not species or tissue specific, or dependent on a single method of purification. The copurifying kinase was stimulated 10-fold by cAMP (10 microM) and 2- to 3-fold by a peptide substrate of the MPC, but was unaffected by protein kinase C activators (calcium and a phospholipid mixture). These findings suggest that protein phosphorylation may represent a mechanism for regulating the activity of the multicatalytic proteinase complex.

A metalloendopeptidase optimally active at a neutral pH was purified 10000-fold from particulate fractions of bovine pituitaries. The solubilized enzyme has an apparent molecular weight of about 90 000, as determined by gel filtration on Sephadex G-200 and G-100 columns. The enzyme is not sensitive to inhibition by SH-blocking agents, diisopropyl fluorophosphate, leupeptin, pepstatin, antipain, and chymostatin. Thiols and metal chelators such as ethylenediaminetetraacetic acid (EDTA) o-phenanthroline are inhibitory. An EDTA-treated enzyme can be reactivated by several divalent metal ions, with zinc giving reactivation at the lowest concentrations. The specificity and kinetic parameters of the enzyme were studied with a series of synthetic peptide naphthylamides. The enzyme cleaves bonds in which the amino group is provided by a hydrophobic amino acid residue (position P1'). Replacement of this residue by small neutral amino acids decreases or virtually eliminates activity. The nature of substituents in positions P1, P2, P3, and P4 greatly influences specificity. Relatively high kcat and kcat/Km ratios were obtained with substrates containing arginine residues in positions P1 and P2. In such cases the impression of a "trypsin-like" activity was created. High reaction rates were also observed with substrates containing small neutral amino acids in positions P1 and P2, provided that position P3 was occupied by the acidic (polar) glutaryl residue. Replacement of this residue with hydrophobic substituents greatly decreased the rate of reaction. When positions P1 and P2, however, were occupied by arginine residues, the unfavorable effect of hydrophobic substituents in position P3 or P4 on catalysis was eliminated.

Pituitary cation-sensitive neutral endopeptidase splits peptide bonds on the carboxyl side of hydrophobic amino acids (chymotrypsin-like activity), basic amino acids (trypsin-like activity), and acidic amino acids (peptidyl-glutamyl-peptide bond hydrolyzing activity). All three activities copurify, are inhibited by cations, and reside in a single high-molecular weight soluble protein complex. Treatment with sodium dodecylsulfate and 2-mercaptoethanol dissociates this complex into five low-molecular weight components. Incubation of the complex at 37 degrees C in buffers of high ionic strength produces aggregation and progressive loss of all three activities. Experiments with inhibitors and activators indicate that the three activities are catalyzed by distinct components. Benzyloxycarbonyl-glycyl-glycyl-leucinal, a peptide aldehyde transition state analog of the substrate used to measure the chymotrypsin-like activity, exclusively inhibits that activity (Ki = 2.5 x 10(-4) M), while markedly activating the trypsin-like activity. The trypsin-like activity is inhibited by leupeptin (Ki = 1.2 x 10(-6) M) and by sulfhydryl blocking agents, and activated by thiols, suggesting that this activity is due to a thiol protease. The peptidylglutamyl-peptide hydrolyzing activity is activated almost 10-fold by low concentrations of sodium dodecylsulfate, inhibited by bovine serum albumin, and suppressed at high enzyme concentrations, suggesting that this component readily interacts with other proteins, including the complex itself. The results indicate that cation-sensitive neutral endopeptidase is a multicatalytic protease complex whose distinct proteolytic activities are associated with separate components of this high-molecular weight protein.

The effect of treatment with L-3,5,3'-triiodothyronine (T3) on the levels of pyroglutamyl peptidase I and pyroglutamyl peptidase II in rat brain regions, pituitary, and serum was studied. Pyroglutamyl peptidase I cleaves pyroglutamyl peptides such as thyrotropin releasing hormone (TRH), luteinizing hormone releasing hormone, neurotensin, and bombesin, whereas pyroglutamyl peptidase II appears to be specific for TRH. Acute administration of T3 did not affect pyroglutamyl peptidase I in any of the regions studied, whereas pyroglutamyl peptidase II was significantly elevated in frontal cortex and pituitary. Treatment with T3 for 10 or 14 days significantly elevated pyroglutamyl peptidase I in pituitary, hypothalamus, olfactory bulb, hippocampus, and thalamus. Chronic T3 treatment elevated pyroglutamyl peptidase II in frontal cortex and in serum. These studies demonstrate regulation of neuropeptide degrading enzymes by thyroid hormones in vivo. This regulation may play a role in the negative feedback control of thyroid status by T3.

The specificity of three neutral endopeptidases toward several biologically active peptides was determined by combined high-performance liquid chromatography and amino acid analysis of the degradation products. Incubation mixtures were chromatographed on a reversed-phase column equilibrated with a mixture of acetonitrile and potassium phosphate buffer (0.05 M; pH 2.0). Reaction products were eluted with a linear gradient of acetonitrile and the absorbance of the effluent monitored at 210 nm. Fractions corresponding to discrete peaks were subjected to quantitative amino acid analysis. The peptide bond undergoing cleavage is readily assigned from the knowledge of the primary structure of the peptide and the amino acid composition of the reaction products.

A highly purified preparation of a cation-sensitive neutral endopeptidase was obtained from bovine pituitaries. The enzyme constitutes almost 0.1% of the protein in bovine pituitary homogenates. Polyacrylamide gel electrophoresis of the enzyme showed a single protein band, and in gel filtration experiments on calibrated Sepharose 6B columns the enzyme eluted slightly ahead of thyroglobulin, suggesting an apparent molecular weight of about 700,000. Polyacrylamide gel electrophoresis in SDS-containing buffers indicated the presence of three major components with molecular weights ranging from about 24,000 to 28,000. The enzyme hydrolyzes bonds between hydrophobic and small neutral amino acids in both model synthetic substrates and biologically active peptides such as substance P, LH-RH, and bradykinin. Peptide bonds in which the carbonyl group is contributed by a glutamyl or arginyl residue are also hydrolyzed, especially if they are preceded in the sequence by hydrophobic amino acids. Leupeptin exclusively inhibited enzymatic activity toward the arginine-containing substrates. This observation, together with the high molecular weight and broad specificity of the enzyme, raised the possibility that the isolated enzyme represents a proteolytic complex composed of units with distinctly different activities. Preliminary attempts to dissociate the enzyme into catalytic units of lower molecular weight were not successful and led to loss of activity.