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)

Previously we reported that ornithine decarboxylase (ODC) is degraded ATP-dependently by the 26 S proteasome in the presence of antizyme (AZ), an ODC inhibitor (Murakami, Y., Matsufuji, S., Kameji, T., Hayashi, S., Igarashi, K., Tamura, T., Tanaka, K., and Ichihara, A. (1992) Nature 360, 597-599). Here we examined the cleavage of ODC by the 26 S proteasome. When ODC purified from ODC-overproducing cells was incubated with the 26 S proteasome and with AZ fused with maltose-binding protein (MBP) in the presence of ATP, ODC was degraded specifically without appreciable breakdown of MBP-AZ. The major degradation products of ODC, which were separated by high performance liquid chromatography on a reverse-phase column, were identified by N-terminal amino acid sequencing. The 26 S proteasome generated a variety of short peptides of 5-11 amino acid residues derived from regions throughout the ODC sequence. No detectable amounts of free amino acid residues were produced, indicating endoproteolytic degradation of ODC by the 26 S proteasome. Their major sites for cleavage of ODC by the 26 S proteasome were on the carboxyl sides of neutral/hydrophobic amino acid residues, but a few were on those of acidic or basic amino acid residues. These results demonstrate that the 26 S proteasome causes exhaustive endoproteolysis of the naturally occurring short-lived protein ODC in a multicatalytic and ATP-dependent manner.
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PMID:ATP- and antizyme-dependent endoproteolysis of ornithine decarboxylase to oligopeptides by the 26 S proteasome. 802 Dec 37

The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs.
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PMID:Multicatalytic proteinase is associated with characteristic oval structures in cortical Lewy bodies: an immunocytochemical study with light and electron microscopy. 802 94

The wild-type tumor suppressor protein p53 is a short-lived protein that plays important roles in regulation of cell cycle, differentiation, and survival. Mutations that inactivate or alter the tumor suppressor activity of the protein seem to be the most common genetic change in human cancer and are frequently associated with changes in its stability. The ubiquitin system has been implicated in the degradation of p53 both in vivo and in vitro. A mutant cell line that harbors a thermolabile ubiquitin-activating enzyme, E1, fails to degrade p53 at the nonpermissive temperature. Studies in cell-free extracts have shown that covalent attachment of ubiquitin to the protein requires the three conjugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubiquitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase, E3. Recognition of p53 by the ligase is facilitated by formation of a complex between the protein and the human papillomavirus (HPV) oncoprotein E6. Therefore, the ligase has been designated E6-associated protein (E6-AP). However, these in vitro studies have not demonstrated that the conjugates serve as essential intermediates in the proteolytic process. In fact, in many cases, conjugation of ubiquitin to the target protein does not signal its degradation. Thus, it is essential to demonstrate that p53-ubiquitin adducts serve as essential proteolytic intermediates and are recognized and degraded by the 26S protease complex, the proteolytic arm of the ubiquitin pathway. In this study, we demonstrate that conjugates of p53 generated in the presence of purified, E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by the 26S protease complex and degraded. In contrast, unconjugated p53 remains stable. The ability to reconstitute the system from purified components will enable detailed analysis of the recognition process and the structural motifs involved in targeting the protein for degradation.
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PMID:Complete reconstitution of conjugation and subsequent degradation of the tumor suppressor protein p53 by purified components of the ubiquitin proteolytic system. 803 27

In the class II region of the major histocompatibility complex (MHC(, four genes implicated in MHC class I-mediated antigen processing have been described. Two genes (TAP1 and TAP2) code for multimembrane-spanning ATP-binding transporter proteins and two genes (LMP2 and LMP7) code for subunits of the proteasome. While TAP1 and TAP2 have been shown to transport antigenic peptides from the cytosol into the endoplasmic reticulum, where the peptides associate with MHC class I molecules, the role of LMP2/7 in antigen presentation is less clear. Using antigen processing mutant T2 cells that lack TAP1/2 and LMP2/7 genes, it was recently shown that expression of TAP1/2 alone was sufficient for processing and presentation of the influenza matrix protein M1 as well as the minor histocompatibility antigen HA-2 by HLA-A2. To understand if presentation of a broader range of viral antigens occurs in the absence of LMP2/7, we transfected T2 cells with TAP1, TAP2 and either of the H-2Kb, Db or Kd genes and tested their ability to present vesicular stomatitis vires and influenza virus antigens to virus-specific cytotoxic T lymphocytes. We found that T2 cells, expressing TAP1/2 gene products, presented all tested viral antigens restricted through either the H-2Kb, Db or Kd class I molecules. We conclude that the proteasome subunits LMP2/7 as well as other gene products in the MHC class II region, except from TAP1/2, are not generally necessary for presentation of a broader panel of viral antigens to cytotoxic T cells. However, the present results do not exclude that LMP2/7 in a more subtle way may, or in rare cases completely, affect processing of antigen for presentation by MHC class I molecules.
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PMID:Presentation of viral antigens restricted by H-2Kb, Db or Kd in proteasome subunit LMP2- and LMP7-deficient cells. 805 44

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

1. Proteins in eukaryotic cells are continually degraded and replaced under precise control mechanisms. Although this continual proteolysis may seem wasteful, it serves several important functions: cells selectively degrade proteins with abnormal sequences or conformations, the accumulation of which could be harmful; the rapid degradation of regulatory peptides and enzymes is essential for the control of metabolic pathways and the cell cycle; and the breakdown of proteins in starvation provides amino acids for gluconeogenesis and energy metabolism. 2. Protein breakdown in eukaryotic cells occurs through distinct pathways: A) lysosomal (involves cathepsins B, H, L, etc.); B) Ca(2+)-dependent (involves Ca(2+)-dependent proteases calpains I and II); C) ATP-dependent, that require or not ubiquitin (comprises at least two large cytosolic proteases, UCDEN and proteasome), and D) ATP-independent (it is not known which proteases are involved in this degradative system). Despite recent dramatic progress, the relative contributions of these pathways to the accelerated proteolysis occurring in normal and pathological states is still largely unknown. 3. In order to identify the cellular mechanisms of skeletal muscle atrophy during fasting and diabetes mellitus, we have studied protein turnover in soleus and EDL muscles from control and fasted (for 24 h) or diabetic rats (1, 3, 5 and 10 days after streptozotocin injection). 4. The increase in muscle proteolysis during fasting seems to be attributable to an enhancement of the energy-requiring process. An increase in the ATP-dependent proteolytic pathway was evident 1 day after food restriction and probably accounted for all of the increased proteolysis demonstrated in the EDL muscles. In parallel with the alterations in the ATP-dependent process, an increase in the ubiquitin-mRNA and proteasome subunit-mRNA was detected. 5. In the acute phase of diabetes (1-3 days) there was an activation of Ca(2+)-dependent (soleus and EDL) and ATP-dependent (EDL) pathways. However, after 5 and 10 days of diabetes the activity of these two pathways fell to values even below control ones. No changes in the lysosomal proteolytic system were observed during diabetes. 6. Although appreciable progress has been made in this research, a large number of important questions remain to be answered, and some of them are discussed in the present paper.
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PMID:Regulation of different proteolytic pathways in skeletal muscle in fasting and diabetes mellitus. 808 98

We demonstrate an essential role for the proteasome complex in two proteolytic processes required for activation of the transcription factor NF-kappa B. The p105 precursor of the p50 subunit of NF-kappa B is processed in vitro by an ATP-dependent process that requires proteasomes and ubiquitin conjugation. The C-terminal region of p105 is rapidly degraded, leaving the N-terminal p50 domain. p105 processing can be blocked in intact cells with inhibitors of the proteasome or in yeast with proteasome mutants. These inhibitors also block the activation of NF-kappa B and the rapid degradation of I kappa B alpha induced by tumor necrosis factor alpha. Thus, the ubiquitin-proteasome pathway functions not only in the complete degradation of polypeptides, but also in the regulated processing of precursors into active proteins.
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PMID:The ubiquitin-proteasome pathway is required for processing the NF-kappa B1 precursor protein and the activation of NF-kappa B. 808 45

In order to identify protein complexes consisting of the proteasome and specific proteasome regulators, crude soluble lysates of red blood cells were fractionated by gel filtration chromatography and by velocity sedimentation centrifugation. The fractionated lysates were then tested for the relative distribution of proteasome activity, proteasome protein, and protein of a known proteasome activator, PA28. At least two proteasome complexes containing PA28 were identified. One of these complexes had an apparent molecular weight of approximately 1,750,000, and appeared to have much more proteasome activity than could be accounted for by its relative concentrations of proteasome and PA28 protein. We hypothesized that this complex contained another activator of the proteasome, and we sought to purify this activator from extracts of red blood cells. A proteasome activator with an apparent molecular weight of approximately 700,000 was identified, purified, and characterized. This activator, termed PA700, greatly stimulated the peptidase activities of the proteasome in an ATP-dependent fashion. PA700 was composed of about 16 polypeptides ranging in molecular weight from 20,000 to 100,000. The ATP-dependent activation of the proteasome by PA700 was closely linked to the formation of a high molecular weight complex that required no additional ATP for activated proteolysis. These results indicate that PA700 is a regulatory protein of the proteasome and is a component of at least one high molecular weight proteasome-containing complex occurring in cell extracts.
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PMID:Identification, purification, and characterization of a high molecular weight, ATP-dependent activator (PA700) of the 20 S proteasome. 810 96

The 26 S proteolytic complex ("26 S proteasome") is a macromolecular assembly thought to be involved in ATP- and ubiquitin-dependent protein degradation in the cytoplasm of higher eukaryotic cells. This complex is composed of one 20 S cylinder particle (multicatalytic proteinase, 20 S proteasome) and two cap-shaped 19 S particles comprising a set of polypeptides in the M(r) range of 35,000-110,000. Here we show that cell supernatant fractions contain both these two subunit complexes as distinct particles as well as assembled to 26 S proteasomes. We have separated and purified all three forms from Xenopus laevis oocytes and have determined their peptidase and protease activities. Using various antibodies specific for either a constitutive p52 polypeptide of the 19 S cap complex or for proteins of the 20 S cylinder particle, we have immunolocalized these complexes in both the cytoplasm and the nucleus of diverse species and cell types. The occurrence of all three forms, the 26 S proteasome, the 20 S cylinder particle, and the 19 S cap complex in the nucleoplasm has also been demonstrated in analyses of isolated giant nuclei from Xenopus oocytes. In addition, we show that the 19 S and 20 S subcomplexes can be released from 26 S proteasomes by ATP depletion and that readdition of ATP to 19 S and 20 S particles in cell extracts leads to the reformation of 26 S proteasomes. We discuss that all three particles (19 S, 20 S, and 26 S) exist in a dynamic equilibrium in both cell compartments and serve cytoplasmic as well as nucleus-specific functions.
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PMID:Distinct 19 S and 20 S subcomplexes of the 26 S proteasome and their distribution in the nucleus and the cytoplasm. 812 97

Proteasomes catalyze the non-lysosomal, ATP-dependent selective breakdown of ubiquitinated proteins and are thought to be responsible for MHC class I-restricted antigen presentation. Recently, we reported that gamma interferon (IFN-gamma) induced not only marked synthesis of the MHC-encoded proteasome subunits LMP2 and LMP7, but also almost complete loss of two unidentified proteasome subunits tentatively designated as X and Y in various human cells. Here, we show that subunit X is a new proteasomal subunit highly homologous to LMP7, and that subunit Y is identical to the LMP2-related proteasomal subunit delta. Thus, IFN-gamma appears to induce subunit replacements of X and Y by LMP7 and LMP2, respectively, producing 'immuno-proteasomes' with the functional diversity responsible for processing of endogenous antigens.
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PMID:Replacement of proteasome subunits X and Y by LMP7 and LMP2 induced by interferon-gamma for acquirement of the functional diversity responsible for antigen processing. 816 24


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