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W Dubiel

Publications and source records attributed to W Dubiel.

At least 37 records · Page 2Linked to original sources
PubMed1997

The 26S proteasome: a dynamic structure.

The proteasomal system consists of a proteolytic core, the 20S proteasome, which associates in ATP-dependent and independent reactions with endogenous regulators providing specific substrate binding sites, chaperone function and regulation of activity to the protease. The best known regulators of the 20S proteasome are the 11S and the 19S complexes. Three subunits of the 20S proteasome and the two subunits of the 11S regulator are induced by gamma-Interferon. However, there are no indications for an influence of gamma-interferon on the subunit composition of the 19S regulator and only a few data exist about the dynamics of this complex. The analysis of 19S regulator subunits from yeast mutants reveals that the ATPases appear to be stringently organized in the 26S complex, while peripheral non-ATPases, such as S5a, might serve as subunits which shuttle substrates to the enzyme. A novel non-ATPase has been cloned, sequenced and identified in a complex besides the 19S regulator, the function of which is presently unknown. The dynamic structure of the 26S proteasome is also characterized by transient associations with components such as the modulator and isopeptidases. Certain viral proteins can also be associated with components of the proteasomal system and alter enzymatic activities.

Humans↗
PubMed1996

Characteristics of 26 S proteases from fission yeast mutants, which arrest in mitosis.

We have isolated the 26 S protease from the fission yeast Schizosaccharomyces pombe. The affinity-purified enzyme contains the two regulatory ATPases mts2+, a homolog of human S4, and CIM5, a homolog of human MSS1 = S7. We show that mts3+, a homolog of the budding yeast NIN1 protein and human S14, is a true component of the 19 S regulatory complex from the fission yeast. The 26 S proteases purified from two thermosensitive mutants, mts2-1 and mts3-1, which arrest in cell cycle at the restrictive temperature (37 degrees C), have been compared with the wild-type enzyme after growing cells at permissive (25 degrees C) and non-permissive temperatures. We demonstrate that mutated mts2 protein is integrated into the protease complex prepared from mts2 cells, whereas mutated mts3 is not present in the 19 S regulatory complex from mts3 cells. The two mutant 26 S proteases isolated after growing cells at 37 degrees C remain stable for two hours at 37 degrees C as measured by ATP-dependent cleavage of the fluorogenic peptide sucLLVY-MCA. At the restrictive temperature, the mutant 26 S proteases do not degrade ubiquitin-[125I]lysozyme conjugates in an ATP-dependent manner, indicating that mts2+ and mts3+ are essential for ubiquitin conjugate degradation. This explains the conditional lethality of the mutants and the cell-cycle arrest in metaphase to anaphase transition. In addition, our data demonstrate that the ATPases of the 26 S enzyme are not redundant.

Adenosine Triphosphate↗
PubMed1995

Molecular cloning and expression of subunit 12: a non-MCP and non-ATPase subunit of the 26 S protease.

A cDNA encoding subunit 12 (S12) of human erythrocyte 26 S protease has been isolated, sequenced and expressed. The cDNA contains an open reading frame that encodes a 36.6 kDA protein 96% identical to mouse Mov-34 and 67% identical to its Drosophila melanogaster homolog. Based on the high degree of sequence identity between human S12, mouse and Drosophila Mov-34 proteins, we conclude that the Mov-34 gene product is a component of the 26 S protease. Antibodies produced against two S12 fragments, Met1-Tyr95 (S12f95) and Met1-Leu205 (S12f205), react with S12 transferred to nitrocellulose from SDS-PAGE. In contrast, after transfer from native gels, the epitope(s) recognized by anti-S12f205 is exposed in the regulatory complex but appears to be masked when the regulatory complex associates with the multicatalytic protease.

Amino Acid Sequence↗
PubMed1995

Identification of a novel ATP-dependent proteolytic activity in mitochondrial intermembrane space.

The formation of primary amines via proteolysis was monitored in isolated rat liver, kidney cortex and heart mitochondria in the presence and in the absence of ATP. The highest proteolytic activity was detected in kidney cortex mitochondria with about 120 nmoles primary amines/hour x mg protein. The formation rates of liver mitochondria amounted to about 100 nmoles primary amines/hour x mg protein and in heart mitochondria about 60 nmoles primary amines/hour x mg protein. In all mitochondria investigated an ATP-dependent proteolysis of 20-40 nmoles primary amines/hour x mg protein was detected. The effects of various protease inhibitors were tested in rat liver mitochondria and thiol-specific reagents showed a 35-70% inhibition. The ATP stimulable portion of proteolysis was blocked by hemin, a known inhibitor of ATP-dependent proteases. The localization of the proteolytic activity was tested by fractionation of the compartments of rat liver mitochondria using the flourogenic peptide suc-Leu-Leu-Val-Tyr-MCA as substrate. About 90% of the ATP-dependent peptide cleavage activity were found in the mitochondrial intermembrane space. The characteristics of the enzyme were compared to those of other known mitochondrial ATP-dependent proteases and it was concluded that it represents a novel proteolytic system of the intermembrane space.

ATP-Dependent Proteases↗
PubMed1994

Molecular cloning and expression of a gamma-interferon-inducible activator of the multicatalytic protease.

The multicatalytic protease (MCP) can be activated by two distinct multisubunit complexes. One is the regulatory component of the 26 S protease, which contains at least 15 distinct subunits. The other is a hexameric activator composed of 31- and 29-kDa subunits. A cDNA for the smaller subunit has been cloned and sequenced. The cDNA encodes a protein of 249 amino acids. Embedded between sequences typical of globular protein domains is a stretch of 28 "alternating" lysine and glutamic acid residues. Similar regions, which we call KEKE motifs, are also found in two MCP subunits, in subunit 12 of the 26 S protease and in a variety of chaperonins including hsp90, hsp70, and calnexin. Expression of the activator cDNA in Escherichia coli produced a functional protein virtually indistinguishable from MCP activator purified directly from red blood cells. The recombinant protein formed three isoelectric species on two-dimensional polyacrylamide gel electrophoresis, and it reacted with antibodies to red blood cell activator. Recombinant activator also bound the multicatalytic protease and stimulated cleavage at the carboxyl terminus of hydrophobic or charged residues. Synthesis of the activator subunit was induced by gamma interferon treatment of HeLa cells. These last two findings have implications for antigen presentation by class I major histocompatibility receptors.

Amino Acid Sequence↗
PubMed1994

Tat-binding protein 7 is a subunit of the 26S protease.

Subunit 6 (S6), an integral component of the 26S protease from human erythrocytes, has been studied by SDS-PAGE, peptide mapping and sequence analysis. S6 was cleaved with CNBr and three internal peptides were sequenced. A comparison with known proteins in Genbank revealed that all three S6 peptides match the predicted sequence of TBP7, Tat-binding protein 7. Based on peptide matches covering more than 10% of the TBP7 sequence, and the fact that the migration of S6 on SDS-PAGE is consistent with the estimated molecular mass for TBP7, we conclude that subunit 6 of the 26S protease is TBP7.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1993

Peptide sequencing identifies MSS1, a modulator of HIV Tat-mediated transactivation, as subunit 7 of the 26 S protease.

Subunit 7 is an integral component of the human erythrocyte 26 S protease. Peptide sequence analysis reveals that 22 amino acids from the N-terminus of subunit 7 correspond exactly to the N-terminus of MSS1, a modulator of HIV gene expression. Additional internal peptides from subunit 7 obtained by CNBr cleavage also match 100% with the deduced amino acid sequence of MSS1. Based on the fact that directly sequenced peptides from subunit 7 are identical to more than 12% of the hypothetical translation product of MSS1, and the fact that the molecular weight of subunit 7 (49 kDa) corresponds to the predicted molecular weight of MSS1 (48,633 Da), we conclude that subunit 7 is MSS1.

ATPases Associated with Diverse Cellular Activitie↗
PubMed1992

Subunit 4 of the 26 S protease is a member of a novel eukaryotic ATPase family.

Ubiquitinated proteins are degraded by a 26 S ATP-dependent protease. SDS-polyacrylamide gel electrophoresis analysis of the purified 26 S enzyme reveals more than 20 polypeptides ranging in apparent molecular masses from 20 to 110 kDa. Although many of the subunits smaller than 30 kDa are members of the multicatalytic protease family, the identity and function of the larger polypeptides have remained unknown. We report here the cDNA sequence for subunit 4, a 51-kDa chain of the 26 S protease. Subunit 4 belongs to a recently identified eukaryotic ATPase family, which includes proteins involved in peroxisome formation, secretion, and human immunodeficiency virus gene expression. Subunit 4 also shows weak similarity to ClpA, the ATP-binding subunit of the Escherichia coli protease, Clp.

Adenosine Triphosphatases↗
PubMed1992

Purification of an 11 S regulator of the multicatalytic protease.

We have identified and purified a protein complex from human red blood cells that activates the multicatalytic protease (MCP). The complex, which we call the regulator, sediments at 11 S and is composed of 30-kDa subunits. The regulator does not hydrolyze fluorogenic peptides, but when multicatalytic protease and regulator are combined, MCP cleaves succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin and Leu-Leu-Glu-p-nitroanilide as much as 60-fold faster. Hydrolysis of several other fluorogenic peptides is stimulated to a lesser extent, and activated MCP does not degrade ubiquitin-lysozyme conjugates, bovine serum albumin, or lysozyme. Latent and activated forms of MCP display similar sensitivity to protease inhibitors, suggesting that activation does not generate new kinds of catalytic sites. In addition, ATP suppresses peptide hydrolysis by activated and latent MCPs to the same extent. Activation involves binding of regulator to MCP, and activated MCP migrates slower on native acrylamide gels. Dissociation of the MCP regulator complex during prolonged sedimentation on glycerol gradients releases active regulator and MCP molecules capable of being reactivated. Moreover, two-dimensional electrophoresis does not reveal changes in MCP or regulator subunits following activation. Thus, activation appears to result from reversible association of regulator subunits with MCP.

Adenosine Triphosphate↗
PubMed1991

Kinetic evidence that the sodium-dependent high-affinity and the sodium-independent low-affinity dopamine uptake are mediated by one carrier.

In synaptosomes of the rat striatum the dopamine uptake was measured in a concentration range of 0.03 microM to 100 microM. In the presence of sodium the uptake exhibited a non-Michaelis-Menten kinetics and in a sodium-free medium the uptake kinetics was sigmoid. According to these findings a novel model for the dopamine uptake is proposed. Its main assumption is one carrier with two dopamine binding sites.

Animals↗
PubMed1991

ATP-dependence of proteolysis in the pulp of rabbit molar teeth.

The ATP-dependent proteolysis of rabbit dental pulp shares a high portion of the total proteolytic capacity in incisor and molar teeth (76 and 69%). It has been found in the fractions of homogenates containing mitochondria, cytosol and microsomes, respectively. The dependence of proteolytic activity on ATP is characterized by an apparent Michaelis constant at cellular levels of ATP (Km = 2.9 mM). The kinetics of inhibition by hemin (Ki = 115.3 microM, n = 2.8) and vanadate (Ki = 1.3 mM, n = 0.93) reveal different modes of actions of both inhibitors. The kinetic features of the ATP-dependent proteolysis in dental pulp differs from those in reticulocytes of the same animal species.

Adenosine Triphosphate↗
PubMed1991

Characterization of a hexammineruthenium-stimulated external NADH oxidase from rat liver mitochondria.

The existence of an external hexammineruthenium-stimulated NADH oxidase in rat liver mitochondria is postulated. This enzyme is localized on the outer surface of the inner mitochondrial membrane, is specific for NADH and requires oxygen. The apparent affinity of the enzyme for NADH amounts to about 4 microM. Furthermore, the enzyme is characterized by an alkaline pH optimum and a linear Arrhenius plot (14 kJ/mol). The electron transfer from NADH to oxygen is not linked with the respiratory chain but is connected with the formation of superoxide radicals.

Animals↗
PubMed1990

The catabolism of endogenous adenine nucleotides in rat liver mitochondria.

The degradation of intramitochondrial adenine nucleotides to nucleosides and bases was investigated by incubating isolated rat liver mitochondria at 37 degrees C under non-phosphorylating conditions in the presence of oligomycin and carboxyatractyloside. Within 30 min the adenine nucleotides were degraded by about 25 per cent. The main products formed were adenosine and inosine the contents of which increased five- to sevenfold. Compartmentation studies revealed that about 50 to 60 per cent of the adenosine formed remained inside the organelles whereas inosine was almost completely released into the surrounding medium. Outside the mitochondria only very small amounts of adenine nucleotides were detected. Similar incubations in the presence of [14C]-adenosine yielded no [14C]-inosine ruling out extramitochondrial adenosine deamination. It is concluded that endogenous adenine nucleotides can be degraded in mitochondria via AMP dephosphorylation and subsequent adenosine deamination. A purine nucleoside transport system mediating at least the efflux of inosine from the mitochondria is suggested.

Adenine Nucleotides↗