Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.25.1 (proteasome)
28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

As an attempt to recruit the third calcium binding site of thermitase into subtilisin BL, a Bacillus lentus alkaline protease (BLAP), the amino acid sequence from position 50 to 60 and position 92 was modified to the equivalent amino acids in thermitase. The resulting protein, designated BLAPm109, exhibited unusual biochemical features. Peptide mapping and gel electrophoresis revealed that two protein species co-purify in a ratio of about 1:1. Form 1 consisted of a single polypeptide of 269 amino acid residues. Form 2 was the same protein but with an internal peptide bond cleavage at the C-terminus of position 54. On electropherograms a dimer of Form 1 and Form 2 was also detectable. A zymogram showed that all three molecular species were catalytically active. From this protein mixture, crystals suitable for X-ray analysis were nevertheless obtained. SDS-PAGE of protein recovered from a crystal revealed that only Form 2 appears. in the crystal. The space group for this crystal was P21 with unit cell dimensions of a=42 angstroms, b=58 angstroms, c=47 angstroms and beta = 106.3 degrees. Examination of the preliminary electron density map revealed that the "thermitase loop" from 50 to 60 departs from the surface of the protein and winds through the active site of a symmetry-related copy of the asymmetric unit.
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PMID:Unusual ligand binding at the active site domain of an engineered mutant of subtilisin BL. 879 30

A ubiquitin(Ub)/ATP-dependent proteolytic complex (26S proteasome) was highly purified from the rat brain. The brain 26S proteasome consisted of 22-110 kDa subunits characteristic of the typical 26S proteasome on the basis of SDS-PAGE. The two-dimensional PAGE (NEPHGE and SDS-PAGE) pattern revealed that the pI values and molecular masses of the brain 26S proteasome subunits were similar to those of the subunits of 26S proteasomes purified from the rat liver and skeletal muscles. The enzymatic properties of the brain 26S proteasome were similar to those of the liver complex and also to the Ub-conjugate degrading activity in the cerebral cortex extract. Furthermore, it was found that the brain 26S proteasome was capable of degrading the myelin basic protein in a Ub-dependent manner. These results indicate that the brain contains the Ub-conjugate-degrading 26S proteasome, the subunit composition of which appears similar to those of the other tissues, and that the myelin basic protein may be a candidate physiological substrate for the brain 26S proteasome.
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PMID:Purification and properties of the 26S proteasome from the rat brain: evidence for its degradation of myelin basic protein in a ubiquitin-dependent manner. 881 59

PA28 is a protein activator of the 20S proteasome. It has a native molecular weight of approximately 200,000 and is composed of six 28,000-dalton subunits arranged in a ring-shaped complex. Purified preparations of PA28 contain two polypeptides, alpha and beta, which are about 50% identical in primary structure. It has been unclear whether native PA28 consists of two distinct homohexameric proteins or of a single protein containing both alpha and beta subunits. To distinguish between these possibilities, we prepared antibodies that reacted specifically with either the alpha or beta subunit and used these subunit-specific antibodies in two types of experiments designed to elucidate PA28 quaternary structure. In the first experiment, the alpha and beta subunits were completely co-immunoprecipitated by each subunit-specific antibody, indicating that both subunits were part of a single protein complex. In the second experiment, PA28 was chemically cross-linked using bis(sulfosuccinimidyl)suberate. When the cross-linked products were immunoblotted after SDS-polyacrylamide gel electrophoresis, indistinguishable patterns were obtained with each subunit-specific antibody. These results confirm that the alpha and beta subunits were part of the same protein complex. The pattern of cross-linked products also provided insight as to the relative abundance and arrangement of the subunits within the PA28 complex and indicated that the ring-shaped PA28 hexamer may be composed of alternating alpha and beta subunits with a stoichiometry of (alphabeta)3. PA28 was inactivated by treatment with carboxypeptidase Y, which cleaved Tyr and Ile residues from the carboxyl terminus of the alpha subunit but had very little effect on the beta subunit. This selective and limited proteolysis prevented binding of both alpha and beta subunits to the proteasome and therefore provides additional evidence of the heterodimeric nature of PA28. These results indicate that a short carboxyl-terminal sequence of the alpha subunit is critical for binding of native PA28 to the proteasome. To learn about the relative functions of the alpha and beta subunits, PA28alpha was expressed in Escherichia coli and purified to homogeneity. Purified PA28alpha stimulated proteasome activity but required 5-10-fold greater concentrations than the heterodimeric PA28 to achieve a given level of activity. These results suggest that the heterodimeric structure of PA28 is required for maximal proteasome activation.
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PMID:A model for the quaternary structure of the proteasome activator PA28. 882 98

The normally labile ornithine decarboxylase (ODC) becomes unusually stable when Cys-441 is replaced with Trp in the variant cell lines HMOA and DH23b. This stable ODC is also observed to have higher mobility on SDS/PAGE. Because previous studies have shown that ODC stability can be achieved when as few as five amino acid residues are removed from its C-terminus, it was suggested that the amino acid substitution in the variant ODC might alter its conformation sufficiently to promote a similar proteolytic loss of a C-terminal degradation signal, resulting in a stable yet active ODC. To examine this mechanism, amino acids in the C-terminal regions of both wild-type and stable (Trp-441) ODC proteins were released, by means of carboxypeptidase-Y digestion, and identified by HPLC. The C-terminal ends were found to be the same, and are as predicted from the cDNA sequence. This study proves that stability of the Trp-441 form of ODC is not simply due to proteolytic removal of a C-terminal proteasome-targeting sequence, thereby implying that the stabilization of this mutant ODC form must result directly from a conformational change associated with the loss of Cys-441.
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PMID:Ornithine decarboxylase stability in HMOA and DH23b cells is not due to post-translational truncation of a C-terminal recognition site. 883 32

LMP-2 and LMP-7, gamma-interferon-inducible subunits of the 20S proteasome, play an important role in antigen processing. To define the molecular basis of their polymorphism, we sequenced Lmp-2 and Lmp-7 cDNA from nine different strains of mice. Three allelic variants of both LMP-2 and LMP-7 were found, but all of the polymorphism in LMP-7 is clustered near the carboxyl terminus of the molecule. We confirmed the nucleotide sequence changes at the protein level in both the unprocessed and processed forms of the molecules by analysis of specific anti-LMP-2, anti-LMP-7 and anti-proteasome immunoprecipitates on two-dimensional PAGE gels. Interestingly, a single amino acid change at position 272 between LMP-7b,d,q and LMP-7k,s,f,x,g7, cas4 from glycine to arginine dramatically affects its migration on SDS-PAGE gels, suggesting the possibility of allele-specific posttranslational modification.
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PMID:Molecular and serological analysis of polymorphisms in the murine major histocompatibility complex-encoded proteasome subunits, LMP-2 and LMP-7. 885 85

A ubiquitin/ATP-dependent proteolytic complex (26S proteasome) was highly purified from rat skeletal muscles and its enzymatic properties were compared with those of the brain 26S proteasome. The purified 26S proteasome comprises 22-110 kDa subunits characteristic of the typical 26S proteasome on the basis of SDS-PAGE. The two-dimensional PAGE (NEPHGE and SDS-PAGE) pattern revealed that the pI values and molecular masses of the muscle 26S proteasome subunits were similar but not identical to those of the subunits of 26S proteasome purified from the rat brain. The enzymatic properties of the muscle 26S proteasome were very similar to those of the brain enzyme in substrate specificity and inhibitor susceptibility. The specific activities of the muscle 26S proteasome toward three fluorogenic peptide substrates were indistinguishable from those of the brain enzyme.
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PMID:Properties of 26S proteasome purified from rat skeletal muscles: comparison with those of 26S proteasome from the rat brain. 886 19

Limited proteolysis of grass carp alcohol dehydrogenase by alkaline protease or subtilisin BPN' at 30 degrees C for 6 hr generated a nicked species that was catalytically active. Electrophoresis on a denaturing SDS-PAGE showed that the 40 kDa subunit of the intact enzyme was cleaved to produce subunits of 27 and 13 kDa, which remained tightly associated with each other under native condition. Such a proteolytically nicked form was catalytically more active than the original intact form of the enzyme. The Vmax value toward the oxidation of ethanol at pH 10 increased by 7.8 fold whereas the K(m) value also exhibited a 140 fold increase. On the other hand, when the same protease treatment was applied to horse liver alcohol dehydrogenase, no activation nor any specific cleavage can be observed.
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PMID:Activation of grass carp liver alcohol dehydrogenase by limited proteolysis. 898 21

Although the structure of the 20 S proteasome from Thermoplasma acidophilum has been elucidated, its enzymatic properties have not been explored in depth. Thermoplasma proteasomes, which contain one type of active site, exhibit not only "chymotrypsin-like" activity (as reported), but also some "post-glutamyl" and "trypsin-like" activities. Like eukaryotic proteasomes, its activity can be stimulated by SDS, Mg2+, and also guanidine HCl, but not urea. The enzyme was strongly inhibited by novel peptide aldehydes with hydrophobic P4 residues, and was rapidly inactivated by 3, 4-dichloroisocoumarin (DCI). DCI modified the N-terminal threonine of the catalytic beta-subunit, the presumed active site nucleophile. To define how proteins are degraded, casein was derivatized with fluorescein isothiocyanate to facilitate detection of released products by the proteasome. Many fluorescent peptides were generated, but the relative amounts of different peptides were independent of the duration of the reaction. The rate of disappearance of protein substrates paralleled the rate of appearance of small products. Unlike conventional proteases, proteasome degrades proteins processively without release of polypeptide intermediates. Upon activation by SDS, guanidine, heat (55 degrees C), or partial inhibition with DCI, proteasomes still functioned processively, but generated a different pattern of peptides under each condition. Thus, processivity is an inherent feature of the 20 S proteasome, not requiring all active sites or ATP hydrolysis.
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PMID:Processive degradation of proteins and other catalytic properties of the proteasome from Thermoplasma acidophilum. 899 62

Seven peptides from subunit 9 (S9) of the human 26S proteasome were sequenced and this information was used to clone a HeLa cDNA that encodes the 46 kDa subunit. Rabbit polyclonal antisera were made against a ubiquitin fusion protein containing 12 amino acids from S9 and against a full-length S9 expressed in E. coli. Western blot analysis showed that the S9-specific antibodies bound the 26S proteasome and its regulatory complex separated on non-denaturing gels. In SDS-PAGE samples of the two complexes, the S9-specific antibodies bound a single 46 kDa subunit. Thus, a cDNA encoding a novel 26S protease subunit has been isolated, sequenced, and expressed.
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PMID:Molecular cloning and expression of subunit 9 of the 26S proteasome. 911 60

Recently, we have reported the isolation and purification of 20S proteasomes from both the procyclic and bloodstream forms of Trypanosoma brucei, but no 26S proteasome was identified under those experimental conditions (Hua et al., Mol. Biochem. Parasitol. (1996) 78, 33-46). Subsequent attempts to identify a 26S proteasome in T. brucei led to the discovery of another form of the 20S proteasome designated the activated 20S proteasome because it exhibited much higher peptidase activities than the original 20S proteasome on all the fluorogenic peptides tested, and it crossreacted with the rabbit antisera against the 20S proteasomes purified from T. brucei. The activated 20S proteasome can be isolated from both procyclic and bloodstream forms of T. brucei and has a slightly higher molecular weight than the 20S proteasome. It is stable in the absence of ATP but susceptible to elution through a DE52 column. Analysis of the activated 20S proteasome in SDS-PAGE showed the presence of all the subunit proteins from the 20S proteasome plus an extra protein with an estimated molecular mass of 26 kDa. This protein, designated PA26, is not a degradation product of the 20S proteasomal subunit proteins. It could be a homolog of the bovine PA28 and human 11S regulator protein which form complexes with the 20S proteasomes resulting in activation of their peptidase activities. This likelihood was confirmed in a reconstitution experiment in which PA26 separated from the proteasome by a DE52 column chromatography was re-introduced into the purified 20S proteasome, and resulted in the emergence of a new protein band with the same mobility and peptidase activities as the activated 20S proteasome in native polyacrylamide gel electrophoresis. The presence of an activated 20S proteasome rather than a homolog of the 26S proteasome in T. brucei suggests that PA26 may play an important role in regulating the proteasome-mediated protein degradations in trypanosomes.
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PMID:Identification and characterization of an activated 20S proteasome in Trypanosoma brucei. 911 74


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