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A J Rivett

Publications and source records attributed to A J Rivett.

At least 37 records · Page 2Linked to original sources
PubMed1995

Degradation of proteasomes by lysosomes in rat liver.

Proteasomes are high-molecular-mass multisubunit complexes which are believed, either by themselves or as a part of the 26S proteinase complex, to play a central role in extralysosomal pathways of intracellular protein breakdown. We have addressed the degradation of proteasomes in rat liver, investigating the possible role of lysosomes. Affinity-purified antibodies against rat liver proteasomes were used for immunoblot analysis of isolated lysosomes. Although proteasomes are not found in lysosomes from normally fed rats, they were found to accumulate in lysosomes of rats treated with leupeptin (an inhibitor of lysosomal proteases) and could also be detected in lysosomes isolated from livers of starved (24 h) rats. Proteinase-K treatment of these fractions, as well as immunogold procedures, show that a proportion of the proteasomes are inside lysosomes. Comparison of the amount of proteasomes found in lysosomes by immunoblotting with their experimentally determined half life (8.3 days) is consistent with an important role of these organelles in the degradation of rat liver proteasomes. Nevertheless, these data do not exclude the possibility that some nonlysosomal degradation of proteasome components also occurs. Since proteasomes were localized in autophagic vacuoles, it is likely that they are taken up mainly by nonselective autophagy. However, using an in vitro system, it was found that, under conditions of starvation, proteasomes may also be taken up into lysosomes and degraded via the heat-shock cognate protein of 73 kDa (hsc73)-mediated transport.

Animals↗
PubMed1995

A pre-existing protease is a common effector of thymocyte apoptosis mediated by diverse stimuli.

Data from a number of model systems support a role for proteolysis in apoptotic cell death. Using immature rat thymocytes, we demonstrate that the inhibitors N-tosyl-L-lysyl chloromethylketone (TLCK) and N-tosyl-L-phenylalanyl chloromethylketone (TPCK) have very different effects on apoptosis. TLCK inhibits apoptosis induced by diverse stimuli at an early stage prior to both DNA fragmentation and cytoplasmic changes. We show that the TLCK-sensitive target is pre-existing and not synthesized in response to apoptotic stimuli. The contrasting effects of TLCK and TPCK support the hypothesis that the TLCK target is a trypsin-like protease which is a common effector of thymocyte apoptosis.

Animals↗
PubMed1995

Catalytic components of proteasomes and the regulation of proteinase activity.

The proteasome (multicatalytic proteinase complex) is a large multimeric complex which is found in the nucleus and cytoplasm of eukaryotic cells. It plays a major role in both ubiquitin-dependent and ubiquitin-independent nonlysosomal pathways of protein degradation. Proteasome subunits are encoded by members of the same gene family and can be divided into two groups based on their similarity to the alpha and beta subunits of the simpler proteasome isolated from Thermoplasma acidophilum. Proteasomes have a cylindrical structure composed of four rings of seven subunits. The 26S form of the proteasome, which is responsible for ubiquitin-dependent proteolysis, contains additional regulatory complexes. Eukaryotic proteasomes have multiple catalytic activities which are catalysed at distinct sites. Since proteasomes are unrelated to other known proteases, there are no clues as to which are the catalytic components from sequence alignments. It has been assumed from studies with yeast mutants that beta-type subunits play a catalytic role. Using a radiolabelled peptidyl chloromethane inhibitor of rat liver proteasomes we have directly identified RC7 as a catalytic component. Interestingly, mutants in Pre1, the yeast homologue of RC7, have already been reported to have defective chymotrypsin-like activity. These results taken together confirm a direct catalytic role for these beta-type subunits. Proteasome activities are sensitive to conformational changes and there are several ways in which proteasome function may be modulated in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. II. Purification and identification of rat A-NKP 1 and A-NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex.

We have recently described nongranular, cytosolic, high-molecular-weight trypsin-like (A-NKP 1) and chymotrypsin-like (A-NKP 2) proteases of interleukin-2-activated rat natural killer (A-NK) cells. A functional correlation between the inactivation of A-NKP 2 and the inhibition of rat A-NK cell-mediated cytotoxicity was found. Herein we describe the 6,000-fold purification of A-NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin-Sepharose chromatography. We also report the novel finding that A-NKP 2 as well as A-NKP 1, derived from either rat A-NK cells or the rat NK leukemic cell line CRNK-16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK-16-derived proteasomal subunits. The identification, characterization, and purification of A-NKP 1 and A-NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A-NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell-mediated cytotoxicity.

Animals↗
PubMed1994

Proteasomes: the changing face of proteolysis.

The proteasome, the multimeric, multicatalytic complex that is responsible for much of the proteolysis within cells, catalyzes several different types of peptide cleavage. Recently, it has been found to increase its repertoire of catalytic activities even further, by varying its catalytic subunits and by associating with regulatory complexes.

Animals↗
PubMed1994

Changes in proteasome localization during the cell cycle.

We have investigated proteasome localization in synchronized cells using polyclonal anti-proteasome antibodies. Proteasomes were localized in the nucleus and cytoplasm at all phases of the cycle, but changes in localization were observed which explain the different immunofluorescence patterns found in asynchronous cells. In the nucleus, the intensity of staining in early S phase was low and showed a punctate distribution which changed to a more diffuse and intense labeling during S to G1. In the cytoplasm, proteasomes were concentrated in the perinuclear region at G1 and at the start of S phase and gradually moved towards the periphery of the cell as the cell cycle progressed to G2. No cell cycle-dependent changes were detected in the rate of synthesis or level of proteasomes. An apparent colocalization of proteasomes with elements of the cytoskeleton mainly observed in G2 was investigated further in PtK2 cells. The overall distribution of proteasomes and cell cycle-dependent changes in PtK2 cells were similar to those in L-132 cells. Double-label immunofluorescence studies using anti-proteasome and anti-cytokeratin (TROMA-1) antibodies showed that proteasomes do colocalize with intermediate filaments of the cytokeratin type, mainly during G2. In mitosis, proteasomes were found by immunogold electron microscopy to be localized around the chromosomes in both PtK2 and L-132 cells. Cell cycle-dependent changes in the localization of proteasomes suggest that they may have a regulatory function related to the cell cycle, for example, in the degradation of proteins which control its progression.

Animals↗
PubMed1993

Controlled high-level expression of the lon gene of Escherichia coli allows overproduction of Lon protease.

Lon protease from Escherichia coli is an ATP-dependent protease which plays important roles in regulating the levels of specific proteins and in eliminating abnormal proteins. A major problem of working with Lon protease, the inability to substantially overproduce the enzyme, has been overcome by placing the lon gene under the control of an inducible trp promoter within a copy-number-controllable plasmid. Induction resulted in higher levels of production of the protease (approximately 100 micrograms/ml of cell culture) than were previously possible. The enzyme has been purified to apparent homogeneity and shown to possess the characteristic ATP-dependent proteolytic activity. Sequence verification during DNA manipulations revealed differences from two previously published sequences for the lon gene.

ATP-Dependent Proteases↗
PubMed1993

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.

Affinity Labels↗
PubMed1993

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.

Animals↗
PubMed1993

cDNA cloning of a new type of subunit of mammalian proteasomes.

The primary structure of a new type of subunit (RN3) of rat proteasomes (multicatalytic proteinase complexes) has been determined from the nucleotide sequence of the cDNA. The cDNA encodes a protein of 232 amino acids but the directly determined N-terminal amino acid sequence suggests that the subunit is post-translationally processed to a M(r) = 24k form. Sequence alignments reveal a similarity of RN3 to other proteasome subunits. It can be designated a B-type proteasomal subunit but is not closely related to the beta subunit of the archaebacterial proteinase or to other members of the B group.

Amino Acid Sequence↗
PubMed1993

Leupeptin-binding site(s) in the mammalian multicatalytic proteinase complex.

The multicatalytic proteinase (MCP) complex is a major nonlysosomal proteinase which plays an important role in non-lysosomal pathways of protein degradation and which has recently been implicated in antigen processing. The mammalian MCP complex is composed of more than 20 different types of polypeptide, but it is not yet clear which of these components are responsible for its proteolytic activities. The complex has at least three distinct types of proteolytic activity. One of these, the so-called 'trypsin-like' activity, which involves cleavage on the carboxy side of basic amino acid residues, can be selectively and completely inhibited by peptidyl arginine aldehydes (such as leupeptin and antipain), and is also the most sensitive to inhibition by thiol-reactive reagents. In the present study N-[ethyl-1-14C]ethylmaleimide has been used to specifically label thiol groups protected by leupeptin binding. The results suggest that one or two polypeptide components within the complex can be protected against modification by N-ethylmaleimide. These components may be responsible for the 'trypsin-like' activity of the complex or may be adjacent to the catalytic component(s) and play an important role in substrate binding.

Amino Acid Sequence↗
PubMed1993

Characterization of proteasomes isolated from rat liver.

Proteasomes are cylindrical particles which have a pseudohelical arrangement of subunits. On 2D-PAGE gels, rat liver proteasome preparations give rise to up to 25 proteins which are encoded by at least 16 different genes that are all members of the same family. Proteasomes are able to degrade protein substrates to acid soluble peptides. They have at least five different catalytic components which can be distinguished by the use of synthetic peptide substrates and inhibitors which have very different reactivity at the different sites. Proteasomes can undergo conformational changes when treated with various effectors of their multiple peptidase activities. They are found in the nucleus and in the cytoplasm and, in cultured cells, show changes in localization during the course of the cell cycle.

Animals↗
PubMed1992

Use of serine-protease inhibitors as probes for the different proteolytic activities of the rat liver multicatalytic proteinase complex.

The multicatalytic proteinase (MCP) complex catalyses cleavage of bonds on the carboxy-group side of basic, hydrophobic or acidic amino acid residues. Originally, it was proposed that the complex contained three distinct types of catalytic component. MCP from rat liver has been assayed for so-called trypsin-like activity with Boc-Leu-Ser-Thr-Arg-NH-Mec (Mec, 4-methylcoumarin; Boc, t-butoxycarbonyl), for chymotrypsin-like activity with Ala-Ala-Phe-NH-Mec and Suc-Leu-Leu-Val-Tyr-NH-MEc (Suc, succinyl), and peptidyl-glutamylpeptide hydrolase activity with Cbz-Leu-Leu-Glu-Nap (Nap, naphthylamide; Cbz, benzyloxycarbonyl). Results of these studies suggest that as many as five distinct components can be distinguished, one for the trypsin-like activity and two for each of the others. The activities were tested with a variety of serine-protease inhibitors, and other novel effectors have also been identified. The two most effective inhibitors were 4-(2-amino-ethyl)benzenesulphonyl fluoride, which selectivity inactivates the trypsin-like activity, and 3,4-dichloroisocoumarin which inhibits chymotrypsin-like activity and the second, cooperative component [Djaballah, H. & Rivett, A. J. (1992) Biochemistry 31, 4133-4141] of peptidylglutamylpeptide hydrolase activity. The three activities inhibited by 3,4-dichloroisocoumarin can easily be distinguished by the effects of chymostatin analogues, diisopropylfluorophosphate, guanidine/HCl and casein. The results support the view that the enzyme is a novel type of serine protease and suggest that it may contain at least five distinct catalytic components. Marked differences in the reactivities of the different catalytic sites with different reagents can be used to distinguish between them.

Amino Acid Sequence↗
PubMed1992

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)

Amino Acid Sequence↗
PubMed1992

Electron microscopic localization of the multicatalytic proteinase complex in rat liver and in cultured cells.

The multicatalytic proteinase (MCP) prosome or proteasome is a large multifunctional complex which is believed to play a major role in non-lysosomal pathways of intracellular protein degradation and has recently been implicated in antigen processing. In this study, affinity-purified antibodies against rat liver MCP were used to investigate the localization of the proteinase both in rat liver and in growing human L-132 cells in culture, using electron microscopic immunogold techniques. Quantitation of the MCP in different subcellular localizations by morphometric analysis of electron micrographs showed the proportion in the nucleus to be 17% for hepatocytes and 51% for L-132 cells, demonstrating differences in the distribution of MCP in different cell types. In hepatocytes, 14% of the total MCP was found associated with the endoplasmic reticulum. The remainder was localized in the cytoplasmic matrix. Immunofluorescence studies with L-132 cells also showed a reaction in nuclei and cytoplasm. The localization of MCP is consistent with its proposed multiple functions in protein turnover, in the production of peptides for antigen presentation, and in RNA processing.

Animals↗