Branched Chain Amino Acid Preferring Research Articles

Peroxynitrite-induced oxidation and its effects on isolated proteasomal systems.

The proteasomes are the major intracellular proteolytic systems involved in the removal of altered proteins. In this study, we examined different susceptibilities of constitutive (XYZ) and interferon-gamma inducible (LMP) 20S proteasomes, isolated from bovine brain and thymus, respectively, to peroxynitrite-mediated oxidation. Exposure of XYZ and LMP proteasomes to increasing amounts of peroxynitrite resulted in different levels, in the two enzymes, of 3-nitrotyrosine groups and tryptophan residues oxidation. 1-Anilino-8-naphtalene-sulfonic acid binding studies and quenching of tryptophan residues indicated that the LMP complex was more sensitive to peroxynitrite. Regarding the proteolytic activities, the XYZ proteasome showed an overall activation (even if the trypsin-like (T-L) component was 20% inhibited), with the peptidyl-glutamyl peptide-hydrolyzing (PGPH) and branched-chain amino acid-preferring (BrAAP) activities being the most stimulated. On the other end, the LMP proteasome was inhibited, especially the BrAAP activity, whereas the T-L activity was not affected. Furthermore, exposure to increasing amounts of peroxynitrite induced a gradual decrease of beta-casein degrading rate by the LMP proteasome, whereas it did not influence the constitutive complex. Our results indicated that peroxynitrite caused a mild modification of the XYZ complex, leading to activation of its catalytic activities. Differently, the LMP proteasome showed a more significant conformational change resulting in the inhibition of the proteolytic functions.

Effect of neurotoxic metal ions on the proteolytic activities of the 20S proteasome from bovine brain.

The effect of oxidative stress induced by neurotoxic metal ions on the properties of the brain 20S proteasome or multicatalytic proteinase complex (MPC) has been studied. Exposure of the 20S proteasome to increasing amounts of Fe(III), Fe(II), Cu(II) or Zn(II) affects its main hydrolytic activities: trypsin-like (T-L), chymotrypsin-like (ChT-L), peptidylglutamyl-peptide hydrolase (PGPH), branched-chain amino acid preferring (BrAAP) and caseinolytic activities, although in different ways. T-L activity showed gradual activation by both iron ions but inhibition by Cu(II) and Zn(II). ChT-L and PGPH activities were inhibited whereas BrAAP activity was widely activated by all the tested metal salts except for zinc ions. Moreover, the exposure to ferrous salt increased the degradation rate of casein. The functional effects appear to be linked to oxidation-induced modifications, as demonstrated by an increase of carbonyl groups following the exposure to metal ions. In addition, modifications induced by ferrous salt on the catalytic subunits were also supported by western blot analyses performed using anti-X, anti-Y and anti-Z antibodies. The results obtained clearly indicate that metal-catalyzed oxidation strongly affects the functions of the brain 20S proteasome, even though the catalytic subunits seem to be differently influenced by oxidative phenomena.

Proteasome from cytokine-treated human cells shows stimulated BrAAP activity and depressed PGPH activity

The branched chain amino acid-preferring (BrAAP) activity of multicatalytic proteinase complex isolated from human umbilical vein endothelial cells and treated with interferon-gamma was increased more than 2-fold, which was associated with a marked increase in LMP7 expression and decreased peptidylglutamyl peptide-hydrolyzing activity. Increases in BrAAP activity in supernatants from cells treated with interferon-gamma, tumor necrosis factor-alpha, interleukin-1 beta, interleukin-6, or lipopolysaccharide paralleled the increases in LMP7 expression. These findings are consistent with the conclusion that the increased BrAAP activity of LMP-containing multicatalytic proteinase complex results from incorporation of LMP7 or other LMP subunits.

Components of the Bovine Pituitary Multicatalytic Proteinase Complex (Proteasome) Cleaving Bonds after Hydrophobic Residues

Two catalytic components of the multicatalytic proteinase complex (MPC, proteasome) designated as chymotrypsin-like (ChT-L) and branched chain amino acid preferring (BrAAP) cleave bonds after hydrophobic amino acids. The possible involvement of the ChT-L and peptidylglutamyl-peptide hydrolyzing (PGPH) activities in the cleavage of bonds attributed to the BrAAP component was examined. Several inhibitors of the ChT-L activity containing a phenylalaninal group did not affect the BrAAP activity at concentrations that were more than 150 times higher than their K(i) values for the ChT-L activity. Concentrations of lactacystin that inactivated more than 90% of the ChT-L activity had no effect on the BrAAP activity. Concentrations of 3,4-dichloroisocoumarin (DCI) that inactivated the ChT-L activity activated by up to 10-fold the BrAAP activity toward synthetic substrates and by more than 2-fold the degradation of the insulin B chain in a reaction not inhibited by Z-LGF-CHO, a selective inhibitor of the ChT-L activity. These findings are incompatible with any significant involvement of the ChT-L activity in the cleavage of BrAAP substrates. Both the native and DCI-treated MPC cleaved the insulin B chain mainly after acidic residues in a reaction inhibited by Z-GPFL-CHO, an inhibitor of the BrAAP and PGPH activities. DCI exposure did not result in acylation of the N-terminal threonine in the active site of the Y subunit. These results suggest involvement of the PGPH activity in the cleavage of BrAAP substrates, but this conclusion is incompatible with DCI activation of the BrAAP activity and inactivation of the PGPH activity, and with the finding that proteins inhibiting the PGPH activity had no effect on the BrAAP activity. Rationalization of these contradictions is discussed.

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans

The claw muscles of large-clawed decapod crustaceans undergo a programmed atrophy in preparation for molting, or ecdysis. This is mediated by five cytosolic proteinases organized into two proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent. The calcium-dependent system consists of four calcium-dependent cysteine proteinases (CDPs I, IIa, IIb, and III; native masses 310, 125, 195, and 59 kDa, respectively) that completely degrade myofibrillar proteins and are activated in atrophic muscles. Immunological analysis shows that the active-site sequence in CDP IIa (60-kDa subunit mass) is similar to that in mammalian CDPs (calpains), and that CDP IIb is homologous to a calpain-like gene isolated from Drosophila cDNA libraries. Increased intracellullar Ca2+ stimulates proteolysis in situ , indicating CDPs play an important role in muscle protein catabolism. The ubiquitin/proteasome-dependent system involves the ATP-dependent conjugation of multi-ubiquitin chains to protein by ubiquitin-conjugating enzymes. This acts as a signal for substrate degradation by the 26S proteasome, a multi-subunit complex consisting of a 20S proteasome catalytic “core” and two PA700 (19S) regulatory complexes. Polyubiquitin mRNA, ubiquitin-protein conjugates, and 20S proteasome are elevated about 5-, 8-, and 2-fold, respectively, during atrophy. A heat-induced form of the 20S proteasome hydrolyzes myosin, troponin, and tropomyosin to large fragments in vitro . Biochemical studies identified the branched-chain amino acid-preferring (BrAAP) activity, one of six distinct catalytic components in the complex, as the activity that carries out these initial cleavages. These results indicate that the ubiquitin/proteasome pathway is involved, but its precise role remains to be resolved.

Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants

Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.

Bovine Spleen Multicatalytic Proteinase Complex (Proteasome): REPLACEMENT OF X, Y, AND Z SUBUNITS BY LMP7, LMP2, AND MECL1 AND CHANGES IN PROPERTIES AND SPECIFICITY

Amino acid sequencing of subunits of the multicatalytic proteinase complex (MPC) isolated from bovine spleen showed an almost complete replacement of the X, Y, and Z subunits, constitutively expressed in most tissues, by the interferon-gamma-inducible LMP7, LMP2, and MECL1 subunits. A comparison with the pituitary MPC found a decreased chymotrypsin-like activity, a depressed peptidylglutamyl-peptide hydrolyzing activity, and a highly active component with properties similar to, but not identical with, that of the pituitary branched chain amino acid preferring (BrAAP) component. Unlike the pituitary BrAAP component, that of the spleen MPC exhibited a greatly decreased Km, a highly increased catalytic efficiency (kcat), and a 80-180 times greater specificity constant (kcat/Km) toward substrates with either branched chain or aromatic amino acid residues in the P1 position. Also, unlike the pituitary BrAAP component, that of the spleen was sensitive to inactivation by 3,4-dichloroisocoumarin and sensitive to inhibition by peptidyl-aldehydes with either phenylalaninal or leucinal residues. Several phenylalaninal peptidyl-aldehydes were identified which selectively inhibited components of the spleen but not of the pituitary MPC. Two of the inhibitors are dipeptidyl-aldehydes, two others are tetrapeptidyl-aldehydes with a Pro residue in the P3 position. The possibility is discussed that the properties and specificity of the spleen MPC are a consequence of the presence of the interferon-gamma-inducible subunits.

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 (α7β7)2. Recent studies identify N-terminal threonines present on some β-subunits as the active-site residues. It has been proposed that the molecule contains three or four proteolytically active subunits [Seemulleret al., Science268, 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 β-casein and inhibits the other catalytic activities.

Specific inhibition of the chymotrypsin-like activity of the proteasome induces a bipolar morphology in neuroblastoma cells.

Lactacystin inhibits cell proliferation and induces a distinctive, predominantly bipolar (two-neurite-bearing) morphology in Neuro 2A murine neuroblastoma cells. It binds with high specificity to the multicatalytic 20S proteasome and inhibits at least three of its peptidase activities (chymotrypsin-like, trypsin-like and peptidylglutamyl-peptide hydrolyzing), each at a different rate, without inhibiting other known proteases. The chymotrypsin-like and trypsin-like activities of the proteasome are inhibited most rapidly, and irreversibly. In an effort to determine which of the peptidase activities needs to be inhibited for neurite outgrowth to occur, we treated Neuro 2A cells with peptide aldehydes that selectively inhibit different proteasome activities. Treatment with peptide aldehydes ending in a hydrophobic residue, all of which inhibit the chymotrypsin-like activity, results in a bipolar morphology in Neuro 2A cells, whereas treatment with a peptide aldehyde inhibitor of the trypsin-like activity does not lead to a detectable change in morphology. One of the inhibitors that induces neurite outgrowth has been previously shown to inhibit the chymotrypsin-like activity of the proteasome without inhibiting the other apparently distinct peptidase activities that cleave after neutral residues, the so-called 'branched chain amino acid preferring' (BrAAP) and 'small neutral amino acid preferring' (SNAAP) activities, or the peptidylglutamyl-peptide hydrolyzing (PGPH) activity. The chymotrypsin-like activity appears to antagonize bipolar-type neurite outgrowth in Neuro 2A cells, while the trypsin-like, PGPH, BrAAP and SNAAP appear not to do so. Selective inhibition of a single peptidase activity, as opposed to general inhibition of the proteasome, appears sufficient to induce a specific cellular process. Selective inhibition might be useful in managing diseases where only one activity is involved without completely inhibiting the proteasome. It is also possible that endogenous regulators of the proteasome could affect cellular processes and that certain peptidase activities of the proteasome may have roles in specifying a given cell fate.

Differential Effects of Bovine PA28 on Six Peptidase Activities of the Lobster Muscle Proteasome (Multicatalytic Proteinase)

PA28, an endogenous activator of the bovine proteasome, stimulated the branched-chain amino acid-preferring (BrAAP; 99-fold activation), small-neutral amino acid-preferring (11-fold), acidic chymotrypsin-like (26-fold), and peptidylglutamyl peptide hydrolase (14-fold) activities of the lobster muscle proteasome, while having little or no effect on the trypsin-like, neutral chymotrypsin-like, and caseinolytic activities. These results show that the BrAAP activity, which has been linked to the degradation of myofibrillar proteins by the heat-activated proteasome, is allosterically regulated. However, the activation by PA28 differs from that induced by heat treatment, since heat activation stimulated both the BrAAP and the proteolytic activities but not the other peptidase activities. PA28 shifted the pH optimum of the acidic chymotrypsin-like activity from pH 6–6.5 to pH 7–7.5, while stimulating the activity about 10-fold. These results suggest that PA28 is involved in the activation of the acidic chymotrypsin-like component at physiological pH.

The Multicatalytic Proteinase (Proteasome) of the Hawkmoth, Manduca sexta: Catalytic Properties and Immunological Comparison with the Lobster Enzyme Complex

The proteasome plays a central role in ubiquitin-dependent and -independent proteolysis in eukaryotic cells, The hawkmoth proteasome was purified from larval body wall and characterized with respect to substrate specificity, sensitivity to protease inhibitors, and cross-reactivity with monoclonal antibodies (mAbs) raised against human placenta proteasome. Leupeptin selectively inhibited the trypsin-like activity (T-L) and N-ethylmaleimide inhibited both T-L and chymotrypsin-like activities, whereas 0.02% sodium dodecyl sulfate stimulated the peptidylglutamyl peptide hydrolase, branched-chain amino acid preferring, and caseinolytic activities 20-, 18-, and 3.8-fold, respectively. All four peptidase activities were inhibited by 3,4-dichloroisocoumarin. One-dimensional immunoblot analysis showed that the level and subunit composition of the proteasome varied between tissues, The relative levels of proteasome were high in intersegmental muscle and ovary, lower in Malpighian tubule, male accessory gland, and ventral nerve cord, and lowest in flight muscle and fat body, The tissues differed in the relative amount of a 41-kDa doublet; a 22-kDa subunit was present only in the male accessory gland, Two-dimensional polyacrylamide gel electrophoresis showed that the hawkmoth proteasome contained at least 26 subunits, compared with 28 subunits in lobster, Immunological analysis using four subunit-specific mAbs identified the putative homologs of the human zeta, C2, C3, and C8 α-type subunits in the hawkmoth and lobster enzymes, Two of the four mAbs reacted with three or more of the hawkmoth subunits and three of the mAbs reacted with two or more of the lobster subunits. In addition, two other mAbs that recognize epitopes shared by a number of α-type subunits indicated that at least 15 (lobster) or 16 (hawkmoth) subunits were α-type. These results suggest that much of the subunit complexity of the arthropod proteasomes is a consequence of extensive post-translational modifications.

Branched-chain-amino-acid-preferring peptidase activity of the lobster multicatalytic proteinase (proteasome) and the degradation of myofibrillar proteins.

The multicatalytic proteinase (MCP or proteasome) is a large proteolytic complex that contains at least five catalytic components: the trypsin-like, chymotrypsin-like, peptidylglutamyl-peptide hydrolase (PGPH), branched-chain-amino-acid-preferring (BrAAP) and small-neutral-amino-acid-preferring activities. We have shown that brief heating of the lobster muscle proteasome activates a proteolytic activity that degrades casein and myofibrillar proteins and is distinct from the trypsin-like, chymotrypsin-like and PGPH components. Here we identify the BrAAP activity as a catalytic component involved in the initial degradation of myofibrillar proteins in vitro. This conclusion is based on the following. (1) The BrAAP component was activated by heat-treatment, whereas the other four peptidase activities were not. (2) The BrAAP and proteolytic activities showed similar sensitivities to cations and protease inhibitors: both were inhibited by 3,4-dichloroisocoumarin, chymostatin, N-ethylmaleimide and Mg2+, but were not affected by leupeptin, phenylmethanesulphonyl fluoride or Li+. (3) The BrAAP activity was inhibited most strongly by casein substrates and troponin; conversely, the troponin-degrading activity was inhibited by the BrAAP substrate. Another significant finding was that incubation of the heat-activated MCP in the presence of chymostatin resulted in the limited cleavage of troponin-T2 (45 kDa) to two fragments of 41 and 42 kDa; this cleavage was completely suppressed by leupeptin. These results suggest that under certain conditions the trypsin-like component can cleave endogenous protein.

Lobster Muscle Proteasome and the Degradation of Myofibrillar Proteins

The lobster proteasome is primarily a cytosolic enzyme in crustacean striated muscles, although a small amount (< 1% of total) occurs in aggregates associated with invaginations of the cell membrane. The complex exists in vitro in three distinct catalytic states (basal, SDS-activated, and heat-activated forms) which have identical subunit compositions. This review summarizes recent results showing that the branched-chain amino acid-preferring (BrAAP) activity mediates the hydrolysis of myofibrillar proteins by the heat-activated proteasome: (a) only the BrAAP activity is stimulated by heat treatment; (b) the BrAAP activity is strongly inhibited by protein substrates, and (c) both the BrAAP and proteolytic activities show similar sensitivities to cations and protease inhibitors.

Catalytic components of the bovine pituitary multicatalytic proteinase complex (proteasome).

The multicatalytic proteinase complex (MPC), also called the proteasome, is a ubiquitous particle (19S) that is required for life. It is found in the cytoplasm and nucleus of all eukaryotic cells where it degrades selected cytosolic and nuclear proteins. It forms the proteolytic core of the 26S complex that represents the final step in the ubiquitin-dependent pathway of proteolysis. The MPC expresses at least five distinct proteolytic activities. Three activities preferring cleavages on the carboxyl side of neutral amino acids were described: an activity cleaving after branched chain residues, termed branched chain amino acid preferring, that is a major factor in the degradation of proteins, an activity preferring cleavages after bulky hydrophobic residues designated chymotrypsin-like, and an activity cleaving between small neutral amino acids. Activities cleaving after basic (trypsin-like) and acidic residues (peptidylglutamyl peptide-hydrolyzing) have also been described. The expression of multiple proteolytic activities with diverse specificities may provide a functional advantage that allows efficient hydrolysis of target proteins.