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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 26 S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins was purified from spinach leaves to near homogeneity by chromatography on DEAE-cellulose, gel filtration on Biogel A-1.5, and glycerol density gradient centrifugation. The purified enzyme was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion coupled with ATPase activity supplying energy for proteolysis and isopeptidase activity to generate free ubiquitin. By nondenaturing electrophoresis, the purified enzyme was separated into two distinct forms of the 26 S complex, named 26 S alpha and 26 S beta proteasomes, with different electrophoretic mobilities. The 26 S proteasome was found to consist of multiple polypeptides with molecular masses of 23-35 and 39-115 kDa, which were thought to be those of a 20 S proteasome with multicatalytic proteinase activity and an associated regulatory part with ATPase and deubiquitinating activities, respectively. The subunit multiplicity of the spinach 26 S proteasome closely resembled that of rat liver with minor differences in certain components. No sulfhydryl bond was involved in the assembly of this multicomponent polypeptide complex. Electron microscopy showed that the 26 S proteasome complex had a "caterpillar"-like shape, consisting of four central protein layers, assumed to be the 20 S proteasome, with asymmetric V-shaped layers at each end. These structural and functional characteristics of the spinach 26 S proteasome showed marked similarity to those of the mammalian 26 S proteasomes reported recently, suggesting that the 26 S proteasome is widely distributed in eukaryotic cells and is of general importance for catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.
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PMID:Purification and characterization of the 26 S proteasome from spinach leaves. 792 95

PA700 is a 700,000-dalton multisubunit protein that activates multiple proteolytic activities of the 20 S proteasome by a mechanism dependent upon ATP hydrolysis (Ma, C.-P., Vu, J.H., Proske, R.J., Slaughter, C.A., and DeMartino, G.N. (1994) J. Biol. Chem. 269, 3539-3547). In order to determine the identities of and structural relationships among the subunits of PA700, individual PA700 subunits were isolated by a combination of reverse phase high performance liquid chromatography (HPLC) and SDS-polyacrylamide gel electrophoresis. Seven of the 16 subunits of PA700 so isolated were subjected to solid phase protease digestion followed by reverse phase HPLC. Selected peptides from each protein were sequenced by automated Edman degradation. Comparison of the resulting amino acid sequences with those in current data bases indicated that three of the subunits represented novel proteins, whereas four subunits were homologous to previously describe proteins. Three subunits of the latter group were, in turn, homologous to one another and are members of a large family of proteins containing a consensus sequence for ATP binding. Purified PA700 demonstrated ATPase activity. Treatment of PA700 with alkylating agents, such as N-ethylmaleimide, inhibited with similar kinetics both proteasome activation and ATPase activity, suggesting that these two activities are functionally linked. Thus, PA700 is composed of multiple members of a protein family that may function in the ATP-dependent regulation of proteasome activity.
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PMID:PA700, an ATP-dependent activator of the 20 S proteasome, is an ATPase containing multiple members of a nucleotide-binding protein family. 806 4

An ATP/ubiquitin-dependent proteasome complex with an apparent sedimentation coefficient of 26S was purified from rat liver to near homogeneity by an improved method based on procedures reported previously. Two electrophoretically distinct forms of the 26S complex, named 26S alpha and 26S beta, with very similar subunit compositions were found not only in purified preparations but also in crude extracts, indicating that the 26S proteasome is present as two isoforms. The 26S proteasome was shown to degrade multi-ubiquitinated, but not unmodified, lysozymes in an ATP-dependent fashion, to have ATPase activity supplying energy for proteolysis, and to contain isopeptidase activity to generate free ubiquitin Mg2+/ATP-dependently. The 26S proteasome also catalyzed the ATP-independent hydrolyses of three types of fluorogenic peptides with basic, neutral, and acidic amino acids at their cleavage sites, respectively. These peptides are also good substrates for the 20S proteasome, but their degradation by the free 20S proteasome and by its assembled form in the 26S complex differ markedly, suggesting a functional difference between the two forms of proteasomes. Electrophoretic and immunochemical analyses showed that the large 26S complex was composed grossly of two different structures: a core 20S proteasome with multicatalytic proteinase functions and an associated part possibly with a regulatory role. These two structures both consisted of multiple polypeptides with molecular masses of 21-31 and 35-110 kDa, respectively. The subunit multiplicity of the rat 26S proteasome closely resembled that of the human counterpart, showing only minor species-specific differences in certain components. The assembly of this multi-component complex was found not to involve a sulfhydryl bond. Electrophoretic peptide mapping with lysyl-endopeptidase indicated the non-identity of the multiple subunits of the 26S proteasome. From these structural and functional characteristics, the 26S proteasome, which is widely distributed in mammals, is suggested to be a new type of multi-molecular complex catalyzing the soluble energy- and ubiquitin-dependent proteolytic pathway.
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PMID:Purification and characterization of the 26S proteasome complex catalyzing ATP-dependent breakdown of ubiquitin-ligated proteins from rat liver. 839 72

A ubiquitin (Ub)/ATP-dependent proteolytic complex (26S proteasome) purified from rabbit skeletal muscle was dissociated into two subcomplexes, a 20S proteasome and a regulatory subunit complex, by preparative non-denaturing polyacrylamide gel electrophoresis (PAGE). The isolated regulatory subunit complex preparation gave a single broad band on analytical non-denaturing PAGE, and several bands ranging between 33 and 110 kDa on SDS-PAGE. This complex was found to consist of about 20 subunits on the basis of two-dimensional PAGE, the pattern of which appeared identical or very similar to that of the 33-110 kDa 26S proteasome subunits. The apparent molecular mass of the complex was estimated to be 1100 kDa by Ferguson plot analysis and also by Superose 6 gel filtration. Unlike the 26S proteasome, neither ATPase activity nor protease activities toward Suc-Leu-Leu-Val-Tyr-MCA, Boc-Phe-Ser-Arg-MCA, Z-Leu-Leu-Glu-beta NA, [14C]-casein, [125I]-lysozyme and Ub-[125I]-lysozyme were significantly detectable in the regulatory subunit complex. This complex was found to be capable of associating with itself in MgATP-dependent manner. These results suggest that a regulatory subunit complex dissociated from the 26S proteasome comprises all the higher molecular mass subunits of the 26S proteasome, and has no detectable ATPase and protease activities, although the homo-oligomerization occurs in an ATP-dependent fashion.
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PMID:Regulatory subunit complex dissociated from 26S proteasome: isolation and characterization. 854 9

The 26S proteasome complex plays a general role in turnover of both short and long lived proteins by specifically degrading ubiquitinated proteins. Recent evidence suggests that this large protease has more specific functions in a number of important cellular processes, ranging from activation of the transcription factor NFkB and antigen processing to transit through mitosis. We have identified a component of the 26S proteasome that interacts specifically with MB67, an orphan member of the nuclear hormone receptor superfamily. MIP224 (MB67 interacting protein) was isolated using the yeast two hybrid system and is apparently identical to the human 26S proteasome component TBP7. MIP224/TBP7 is one of several proteasomal proteins that share a strongly conserved ATPase domain (CAD) which is also present in a rapidly expanding superfamily of proteins with diverse functions. In yeast, MIP224 interacts specifically with MB67 and another closely related orphan receptor, but does not interact with several other receptor superfamily members tested. In mammalian cells, coexpression of MIP224 inhibits transactivation by MB67. MIP224 also interacts in yeast with other CAD proteins, including MSS1, which is proteasomal, and TRIP1, which is associated with transcriptional activation. This interaction of a proteasomal protein with a transcriptional protein suggests a previously unexpected link between the processes of protein degradation and transcriptional regulation.
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PMID:A component of the 26S proteasome binds on orphan member of the nuclear hormone receptor superfamily. 860 43

The activity of the intracellular protease, the proteasome, is modulated by a number of specific regulatory proteins. One such regulator, PA700, is a 700,000-Da multisubunit protein that activates hydrolytic activities of the proteasome via a mechanism that involves the ATP-dependent formation of a proteasome-PA700 complex. Four subunits of PA700 have been shown previously to be members of a protein family that contains a consensus sequence for ATP binding, and purified PA700 expresses ATPase activity. We report here the identification, purification, and initial characterization of a new modulator of the proteasome. The modulator has no direct effect on the activity of the proteasome, but enhances PA700 activation of the proteasome by up to 8-fold. This activation is associated with the formation of a proteasome/PA700-containing complex that is significantly larger than that formed in its absence. The modulator has a native Mr of approximately 300,000, as determined by gel filtration chromatography, and is composed of three electrophoretically distinct subunits with Mr values of 50,000, 42,000, and 27,000 (p50, p42, and p27, respectively). Amino acid sequence analysis of the subunits shows that p50 and p42 are members of the same ATP-binding protein family found in PA700. The p50 subunit is identical to TBP1, a protein previously reported to interact with human immunodeficiency virus Tat protein (Nelbock, P., Dillion, P. J., Perkins, A., and Rosen, C. A. (1990) Science 248, 1650-1653), while the p42 subunit seems to be a new member of the family. The p27 subunit has no significant sequence similarity to any previously described protein. Both p50 and p42, but not p27, were also identified as components of PA700, increasing the number of ATP-binding protein family members in this complex to six. Thus, p50 and p42 are subunits common to two protein complexes that regulate the proteasome. The PA700-dependent proteasome activator represents a new member of a growing list of proteins that regulate proteasome activity.
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PMID:Identification, purification, and characterization of a PA700-dependent activator of the proteasome. 862 9

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.
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PMID:HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. 865 Jan 74

Molecular cloning of cDNA for a new regulatory subunit, designated p97, of the human 26S proteasome showed that the polypeptide consists of 908 amino acid residues with a calculated molecular mass of 100184 Da and an isoelectric point of 4.94. Computer analysis showed that p97 is very similar to type-1 tumor-necrosis-factor (TNF)-receptor-associated protein (TRAP)-2 and 55.11, both of which were identified recently as binding proteins of the cytoplasmic domain of type-1 TNF receptor by yeast two-hybrid screening. This finding suggests that the 26S proteasome might serve as a mediator molecule in the TNF signaling pathway in cells. Computer-assisted similarity analysis also revealed the high sequence similarity of p97 with a yeast protein whose function is yet unknown, the gene for which is here termed NAS1 (non-ATPase subunit 1). Disruption of NAS1 resulted in several phenotypes, including lethality and temperature-sensitive growth, depending on the genetic background of the cells used. The human p97 cDNA suppressed the growth defect of nas1 disruptant cells, when expressed from single-copy or multi-copy vectors, indicating that p97 is functionally equivalent to yeast Nas1p. Culturing of the temperature-sensitive nas1 cells at the restrictive temperature promoted the accumulation polyubiquitinated cellular proteins, implying that the 26S proteasome requires a functional Nas1p subunit for ubiquitin-dependent proteolysis. These results indicate that p97/Nas1p plays an important regulatory role in the function of the 26S proteasome.
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PMID:cDNA cloning and functional analysis of the p97 subunit of the 26S proteasome, a polypeptide identical to the type-1 tumor-necrosis-factor-receptor-associated protein-2/55.11. 877 43

The ocular lens consists of a single layer of epithelial cells on its anterior surface and underlying fiber cells, which are derived from the epithelial cells by differentiation and make up the bulk of the lens. Because lens cells are segregated by age and stage of differentiation, we are using this tissue to study the role of the proteasome in differentiation. The purpose of this study is to corroborate the ATPase function of chick subunit 4 (cS4) and assess the levels of the mRNA in the differentiating lens relative to other tissues. We have generated a computer model of the tertiary structure of the ATPase domain of the cS4 of the ATPase complex that regulates the 20S proteasome. The predicted polypeptide from the cloned cDNA of cS4 (440 residues) had a calculated molecular mass of 49,182 and is 98 and 73% identical to human and yeast S4 protein sequences, respectively. A computer search for comparison with known proteins in GenBank showed that the cS4 protein sequence has a conserved region of about 200 amino acid residues including an ATP/GTP binding site and a mitochondrial energy transfer proteins signature sequence. Based on secondary structure, the computer-generated model of the ATPase domain is comparable to that of RecA, with a root mean square deviation of 0.851 from the RecA triad. mRNA in the 14-day-old chick embryo lens is derived primarily (90%) from differentiating cells. The level of cS4 mRNA determined by quantitative RT/PCR in this differentiating tissue was comparable to the cS4 mRNA levels in chick liver, heart, and brain.
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PMID:cDNA cloning of a chick homologue of human ATPase complex subunit 4, quantitative tissue distribution and tertiary structure comparison of the ATPase domain to RecA. 880 79

Previously, we have shown extensive reprogramming of the ATPase regulator of the 26S proteasome preceding the programmed destruction of intersegmental muscles (ISM) in the tabacco horn moth Manduca sexta (Dawson et al., J. Biol. Chem, 270, 1850-1858, 1995). We now show that the extensive reprogramming of the regulatory components of the 26S proteasome occurs only in ISM and not in flight muscles (FM), which undergo terminal differentiation at ecdysis. Unlike in ISM, the ATPase regulators, MS73, MSS1, TBP1 and mts2, remain at low levels in 26S proteasomes in FM from developmental Stage-0 to Stage-7. The non-ATPase regulator subunit 5a, which binds to multiubiquitin chains, increased in ISM similarly to the ATPases but not in FM. The ecdysteroid agonist RH-5849 prevented these subunit increases in ISM. These findings show that reprogramming of 26 S proteasomes is involved in the specific elimination of ISM during eclosion and does not occur in FM which are needed for adult moth flight.
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PMID:Specific developmental changes in the regulatory subunits of the 26 S proteasome in intersegmental muscles preceding eclosion in Manduca sexta. 892 Sep 45


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