Peptidylglutamyl-peptide Hydrolase Research Articles

Cloning and expression of a human pro(tea)some β-subunit cDNA: A homologue of the yeast PRE4-subunit essential for peptidylglutamyl-peptide hydrolase activity

The cDNA encoding a human prosome β-subunit (HSBpros26) was isolated from a lymphoma library using the cDNA of the Xenopus homologue as a probe. The cDNA contains an open reading frame encoding a protein of 233 amino acids and a calculated molecular weight of 25,909. Comparison with interspecies homologues of HSBpros26 from Xenopus (XLB), rat (RN3) and yeast (PRE4) reveals a high degree of identity between the β-subunits except for the N-terminal end, which is probably cleaved post-translationally. The complete coding sequence of HSBpros26 has been expressed in E. coli. The produced protein of about 27 kDa reacts with the prosomal monoclonal antibody MCP205, kindly provided by Dr. K. Hendil. The molecular weight of the native protein is about 28 kDa indicating that the protein is present as monomers. Finally partially purified HSBpros26 preparations do not contain any proteolytical activity.

Reaction of proteasomes with peptidylchloromethanes and peptidyldiazomethanes.

The multicatalytic endopeptidase complex (proteasome) has multiple distinct peptidase activities. These activities have often been referred to as 'chymotrypsin-like', 'trypsin-like' and 'peptidylglutamyl-peptide hydrolase' activities according to the type of residue in the P1 position, although it is now clear that mammalian proteasomes have at least five distinct catalytic sites. In the present study, potential affinity-labelling reagents (peptidylchloromethanes, peptidyldiazomethanes, a peptidylfluoromethane and peptidylsulphonium salts) containing hydrophobic, basic or acidic amino acid residues in the P1 position have been tested for inhibition of the different activities of the rat liver proteinase complex. The results show that individual peptidase activities of proteasomes can be inhibited by a variety of peptidylchloromethanes and peptidyldiazomethanes. Although the rate of inactivation of proteasomes by even the most effective peptidylchloromethanes and peptidyldiazomethanes are often quite slow (k(obs)/[I] in the range 0.1-10 M-1 x s-1) compared with the reaction of similar compounds with some other proteinases, the results provide useful information concerning the specificity of the distinct catalytic centres of proteasomes, and some selective affinity-labelling reagents have been identified. Tyr-Gly-Arg-chloromethane was found to be a useful inhibitor of trypsin-like activity. Inhibition of the other peptidase activities was often incomplete, even after repeated addition of inhibitor, and it proved to be difficult to predict the effect of different reagents. For example, Cbz-Tyr-Ala-Glu-chloromethane was found to inhibit 'chymotrypsin-like' activity (assayed with Ala-Ala-Phe-7-amino-4-methylcoumarin or succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin), while the best inhibitors of 'peptidylglutamyl-peptide hydrolase' activities (assayed with benzyloxycarbonyl-Leu-Leu-Glu beta-naphthylamide) were peptidyldiazomethanes containing hydrophobic amino acid residues. These results suggest that the original nomenclature of proteasome activities is misleading, because the residue in the P1 position is not the only determinant of specificity.

The multicatalytic proteinase complex (proteasome): structure and conformational changes associated with changes in proteolytic activity

The multicatalytic proteinase complex or proteasome is a high-molecular-mass multisubunit proteinase which is found in the nucleus and cytoplasm of eukaryotic cells. Electron microscopy of negatively stained rat liver proteinase preparations suggests that the particle has a hollow cylindrical shape (approximate width 11 nm and height 17 nm using methylamine tungstate as the negative stain) with a pseudo-helical arrangement of subunits rather than the directly stacked arrangement suggested previously. The side-on view has a 2-fold rotational symmetry, while end-on there appears to be six or seven subunits around the ring. This model is very different from that proposed by others for the proteinase from rat liver but resembles the structure of the simpler archaebacterial proteasome. The possibility of conformational changes associated with the addition of effectors of proteolytic activity has been investigated by sedimentation velocity analysis and dynamic light-scattering measurements. The results provide the first direct evidence for conformational changes associated with the observed positive co-operativity in one component of the peptidylglutamylpeptide hydrolase activity as well as with the stimulation of peptidylglutamylpeptide hydrolase activities by MnCl2. In the latter case, there appears to be a correlation between changes in the shape of the molecule and the effect on activity. KCl and low concentrations of SDS may also act by inducing conformational changes within the complex. Sedimentation-velocity measurements also provide evidence for the formation of intermediates during dissociation of the complex by urea, guanidinium chloride or sodium thiocyanate. Dissociation of the complex either by these agents or by treatment at low pH leads to inactivation of its proteolytic components. The results suggest that activation and inhibition of the various proteolytic activities may be mediated by measurable changes in size and shape of the molecules.

Peptidylglutamyl-peptide hydrolase activity of the multicatalytic proteinase complex: evidence for a new high-affinity site, analysis of cooperative kinetics, and the effect of manganese ions.

The multicatalytic proteinase (MCP) complex or proteasome is a major nonlysosomal proteinase of eukaryotic cells. The proteinase can cleave peptide bonds on the carboxyl side of hydrophobic, basic, or acidic amino acid residues. These activities have been referred to as "chymotrypsin-like", "trypsin-like", and "peptidylglutamyl-peptide hydrolase" activities, respectively, and have been shown to be catalyzed at distinct sites. The latter activity is often assayed with the synthetic peptide substrate Z-Leu-Leu-Glu-beta-naphthylamide (LLE-NA). N-tBoc-Ala-Ala-Asp-SBzl is also a substrate for the rat liver MCP, suggesting a broader specificity for cleavage on the carboxyl side of acidic residues than the peptidylglutamyl-peptide hydrolase activity previously reported. The pH optimum is in the range of pH 7.0-7.5. Studies of the dependence of velocity on LLE-NA concentration show (a) that there is a high-affinity site (LLE1) which obeys Michaelis-Menten kinetics with a Km value of approximately 100 microM and (b) that at higher substrate concentrations (LLE2) the curve is sigmoidal, suggesting either allosteric activation of the proteinase at a second site or the involvement of multiple catalytic sites which display positive cooperativity. Activity at the high-affinity site (LLE1) can be distinguished from that of the activity of the LLE2 component by the effect of inhibitors, divalent metal ions, and KCl, as well as by its response to heat treatment. The addition of 1 mM MnCl2 stimulates both LLE1 and LLE2 activities and also permits saturation of MCP with substrate at concentrations of LLE-NA below the solubility limit of this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Sodium dodecyl sulfate and heat induce two distinct forms of lobster muscle multicatalytic proteinase: The heat-activated form degrades myofibrillar proteins

A multicatalytic proteinase (MCP) purified from lobster claw and abdominal muscles degrades a variety of peptide and protein substrates. The enzyme is activated by low concentrations (0.03%) of sodium dodecyl sulfate (SDS) and brief (1 min) heating at 60 °C. The lobster MCP can assume three stable and functionally distinct states in vitro; these are classified as the basal, heat-activated, and SDS-activated forms. The basal MCP possessed high trypsin-like peptidase activity and low chymotrypsin-like peptidase, peptidylglutamyl-peptide hydrolase, and caseinolytic activities; incubation of the basal form with SDS stimulated the peptidylglutamyl-hydrolase activity about 30-fold and inhibited the other three activities 80% to 100%. Heating the basal form stimulated caseinolytic activity about 6-fold with little effect on the peptidase activities. The heat-activated enzyme also degraded myosin, tropomyosin, troponin, and actin depolymerizing factor; α-actinin was resistant to proteolysis. Incubation of the heat-activated MCP with SDS inhibited the trypsin-like, chymotrypsin-like, and proteinase activities 95 to 100% and stimulated the peptidylglutamyl-hydrolase activity about 16-fold. Incubation of myosin with either the basal or the heat-activated forms in the presence of SDS generated identical proteolytic fragments of the myosin heavy chain, suggesting that SDS induced a third form that can be produced from either the basal or the heat-activated forms. The heat-activated form produced proteolytic fragments of myosin heavy chain different from those generated by either basal or heat-activated enzymes in the presence of SDS. Furthermore, 100 m m KCl stimulated the caseinolytic activity of the heat-activated form 24% and inhibited the trypsin-like and peptidylglutamyl-hydrolase activities 56 and 20%, respectively. These results, though indirect, suggest that heating induced a proteinase activity that was distinct from the three peptidase activities. Activation of the basal form with SDS was reversible, since precipitation of dodecyl sulfate with 100 m m KCl restored trypsin-like activity and inhibited peptidylglutamyl-hydrolase activity. In contrast, removal of dodecyl sulfate from the SDS-activated form that was derived from the heat-activated MCP induced its conversion to the basal form. Thus, although heat-activation was irreversible, the heat-activated form was converted back to the basal form via the SDS-activated form.

Catalytic subunits of the multicatalytic proteinase

The multicatalytic proteinase (MCP) has been found in a wide variety of eukaryotic cells [1,2], and a similar enzyme has been identified in archaebacteria [ 3 ] . MCP is a high molecular mass (700 kDa) non-lysosomal proteinase which may be involved in both ubiquitin-dependent and ubiquitin-independent pathways of intracellular protein degradation. The proteinase complex isolated from rat liver seems to be composed of approximately 20 different components with molecular masses ranging from 22 to 34 kDa and isoelectric points ranging from 5 to 8.5 [41. The subunits appear by electron microscopy to be arranged in the shape of a hollow cylinder. The proteinase complex can cleave peptide bonds on the carboxyl side of basic, hydrophobic and acidic amino acid residues [5], and the proteolytic activities have been referred to as trypsin-like, chymotrypsin-like and peptidyl glutamyl peptide hydrolase activities, respectively. Results of studies with a variety of inhibitors have shown that these activities are catalysed at distinct sites. It is of interest to identify which components of the complex are responsible for each of the different proteolytic activities. In this study a method has been devised for selectively labelling MCP components responsible for the trypsin-like activity. The proteinase complex was isolated from fresh rat livers using a series of chromatographic steps [4]. The method of labelling involves use of a site-specific reversible inhibitor of MCP (leupeptin) and a group-specific irreversible inhibitor (N-ethylmaleimide) . Leupeptin inhibits serine and cysteine proteinases by forming a hemiacetal or thiohemiacetal with the catalytic serine or cysteine residue, respectively [7]. Leupeptin selectively inhibits the trypsin-like activity of the MCP complex in a competitive fashion at concentrations which have no effect on the other proteolytic activities. This inhibition can be reversed either by dialysis or by the addition of sodium borohydride to reduce the reactive aldehyde group. However, the recovery of activity by either of these methods was found to be slow. NEM is also an inhibitor of the trypsinlike activity of MCP [El. It also inhibits the peptidyl glutamyl peptide hydrolase activity of MCP but has little effect on the chymotrypsin-like activity. NEM is an irreversible and specific modifier of thiol groups at neutral pH, and is available in a [14C] -labelled form. Leupeptin and NEM were used together in a differential labelling protocol where MCP was first protected by leupeptin and then modified by NEM. Both inhibitors were used at concentrations which were known to fully inhibit the trypsin-like activity. Leupeptin was removed by dialysis and then [14C]NEM was added to label sites which were previously protected by leupeptin. An experiment was also carried out in the absence of leupeptin using only labelled NEM to establish how many thiol groups react with NEM. After dialysis to remove excess labelled NEM, polypeptide components of the two labelled proteinase preparations were separated by two-dimensional SDS polyacrylamide gel electrophoresis. Labelled component (s) were identified by fluorography of the dried gel. The results of these experiments showed that up to 28 moles of [l4C1NEM were incorporated in to each mole of unprotected MCP, whereas only approximately two moles of [I4C]NEM were incorporated per mole of MCP following the differential labelling protocol. These results imply protection by leupeptin of two thiol groups per MCP molecule. Fluorography of the two different labelled MCP preparations showed that almost every component of the complex can be labelled by NEM, but that in experiments designed to label only those NEM-reactive thiol groups protected by leupeptin, only one component is strongly labelled. These labelling experiments suggest that the trypsin-like activity is located in only one of the many different components of the proteinase complex. Recent results from the labelling of catalytic sites possessing the chymotrypsin-like activity also suggest that only one component is involved [9]. These findings support the view that MCP is a complex composed of distinct components which have different functions. This work was supported by the Medical Research Council. A.J.R. is a Lister Institute Research Fellow.