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)

Using specific substrates, benzyloxycarbonyl-Gly-Gly-Leu-p-nitroanilide, benzyloxycarbonyl-Gly-Gly-Arg-2-naphthylamide and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide, cytosolic chymotrypsin-like, trypsin-like and cucumsin-like activities were determined, respectively, in rat epithelial tissues and differentiated human Caco-2 cells. The cytosolic fractions of rat colonic, rectal, nasal, and alveolar epithelial cells and differentiated human Caco-2 cells contained these three distinct enzyme activities. However, effects of enzyme inhibitors revealed that these three distinctive activities were not extensively involved in cytosolic or homogenate degradation of insulin and insulin-like growth factor I (IGF-I). It is concluded that proteasome-like activities may not significantly limit nonparenteral absorption of peptide and protein drugs such as insulin and IGF-I.
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PMID:The involvement of cytosolic chymotrypsin-like, trypsin-like, and cucumsin-like activities in degradation of insulin and insulin-like growth factor I by epithelial tissues. 858 71

The proteasome is responsible for the non-lysosomal degradation of misfolded, transient, or ubiquitin-tagged proteins. This fact and the identification of two major-histocompatibility-complex-(MHC)-encoded proteasomal subunits, LMP2/7, suggest an important role of the proteasome in antigen processing. Using purified 20S proteasomes from a wild-type and a LMP2/7-deletion T lymphoblastoid cell line, we analyzed the effect of LMP2/7 on the peptidase and proteolytic activities of the complex in the context of various purification and activation methods. The incorporation of LMP2/7 alters the peptidase activity against fluorogenic substrates, but these effects are not reflected in the time-dependent degradation pattern of oxidized insulin B chain or of peptide epitopes of an antigenic protein. No effect of LMP2/7 on the degradation pattern of these substrates was observed by either reverse-phase chromatography, pool sequencing, or mass spectrometry. The 20S proteasome can cleave insulin B chain at nearly every position, showing that the P1 position alone does not determine the cleavage sites. The maximum of the length distribution of the end products, makes these ideal candidates for MHC display; yet we find that a natural epitope derived from human histone H3 is further degraded by 20S proteasomes. Alanine scans and substitutions with related amino acids of this epitope indicate that, as in insulin B chain, the cleavage sites are not determined by the P1 position alone.
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PMID:Effects of major-histocompatibility-complex-encoded subunits on the peptidase and proteolytic activities of human 20S proteasomes. Cleavage of proteins and antigenic peptides. 863 60

In normal subjects and diabetic patients, insulin suppresses whole body proteolysis suggesting that the loss of lean body mass and muscle wasting in insulinopenia is related to increased muscle protein degradation. To document how insulinopenia affects organ weights and to identify the pathway for accelerated proteolysis in muscle, streptozotocin-treated and vehicle-injected, pair-fed control rats were studied. The weights of liver, adipose tissue, and muscle were decreased while muscle protein degradation was increased 75% by insulinopenia. This proteolytic response was not eliminated by blocking lysosomal function and calcium-dependent proteases at 7 or 3 d after streptozotocin. When ATP synthesis in muscle was inhibited, the rates of proteolysis were reduced to the same level in insulinopenic and control rats suggesting that the ATP-dependent, ubiquitin-proteasome pathway is activated. Additional evidence for activation of this pathway in muscle includes: (a) an inhibitor of proteasome activity eliminated the increased protein degradation; (b) mRNAs encoding ubiquitin and proteasome subunits were increased two- to threefold; and (c) there was increased transcription of the ubiquitin gene. We conclude that the mechanism for muscle protein wasting in insulinopenia includes activation of the ubiquitin-proteasome pathway with increased expression of the ubiquitin gene.
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PMID:Muscle wasting in insulinopenic rats results from activation of the ATP-dependent, ubiquitin-proteasome proteolytic pathway by a mechanism including gene transcription. 887 19

Upon fasting, the ubiquitin-dependent proteolytic system is activated in skeletal muscle in parallel with the increases in rates of proteolysis. Levels of mRNA encoding the 14 kDa ubiquitin-conjugating enzyme (E2(14K)), which can catalyse the first irreversible reaction in this pathway, rise and fall in parallel with the rates of proteolysis [Wing and Banville (1994) Am.J. Physiol. 267, E39-E48], indicating that the conjugation of ubiquitin to proteins is a regulated step. To characterize the mechanisms of this regulation, we have examined the effects of insulin, insulin-like growth factor I (IGF-I) and des(1-3) insulin-like growth factor I (DES-IGF-I), which does not bind IGF-binding proteins, on E2(14K) mRNA levels in L6 myotubes. Insulin suppressed levels of E2(14K) mRNA with an IC50 of 4 x 10(-9) M, but had no effects on mRNAs encoding polyubiquitin and proteasome subunits C2 and C8, which, like E2(14K), also increase in skeletal muscle upon fasting. Reduction of E2(14K) mRNA levels was more sensitive to IGF-I with an IC50 of approx. 5 x 10(-10) M. During the incubation of these cells for 12 h there was significant secretion of IGF-I-binding proteins into the medium. DES-IGF-I, which has markedly reduced affinity for these binding proteins, was found to potently reduce E2(14K) mRNA levels with an IC50 of 3 x 10(-11) M. DES-IGF-I did not alter rates of transcription of the E2(14K) gene, but enhanced the rate of degradation of the 1.2 kb mRNA transcript. The half-life of the 1.2 kb transcript was approximately one-third that of the 1.8 kb transcript and can explain the more marked regulation of this transcript observed previously. This indicates that the additional 3' non-coding sequence in the 1.8 kb transcript confers stability. These observations suggest that IGF-I is an important regulator of E2(14K) expression and demonstrate, for the first time, stimulation of degradation of a specific mRNA transcript by this hormone, while overall RNA accumulates.
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PMID:Insulin-like growth factor I stimulates degradation of an mRNA transcript encoding the 14 kDa ubiquitin-conjugating enzyme. 891 81

Insulin-degrading enzyme (IDE) is a component of a cytosolic complex that includes multicatalytic proteinase (MCP), the major cytoplasmic proteolytic activity. Insulin, the primary substrate for IDE, inhibits the proteolytic activity of the IDE-MCP complex but not of purified MCP. This provides a regulatory role for IDE in cellular proteolysis and a potential mechanism for intracellular insulin action. To examine the specificity and to explore the mechanisms for the IDE-MCP interaction, we studied the functional interaction of a variety of peptides with the complex. Atrial natriuretic peptide (ANP), relaxin, glucagon, proinsulin, and insulin-like growth factor II (IGF-II) bind to and are degraded by IDE. These peptides have significant inhibitory effects on the chymotrypsin-like and trypsin-like MCP catalytic activities but not the peptidyl-glutamyl hydrolyzing activity. A panel of peptides that are not ligands of IDE had no effect. To explore the potential mechanism for the IDE control of MCP activity, dose response curves for insulin-like growth factor I (IGF-I) and IGF-II effects on MCP chymotrypsin-like activity were determined. IGF-II, which (similar to insulin) is a good substrate for IDE, had a substantial inhibitory effect, whereas IGF-I, which is bound but poorly degraded, had little inhibitory activity on MCP. Proinsulin, another ligand of IDE that is tightly bound but poorly degraded, had a partial effect on MCP activity, but inhibited the full insulin effect. These data suggest a requirement for both the binding and degradation of IDE ligands for the full inhibition of MCP. Insulin-sized degradation products, substrates of IDE, also inhibited MCP activity. Further examination of the insulin effect on MCP included kinetic studies. Insulin produced a noncompetitive inhibition of both the chymotrypsin-like and trypsin-like activities of MCP. These data suggest that the insulin-IDE effect on MCP is due to conformational changes in the IDE-MCP complex and provide an intracellular mechanism of action for insulin.
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PMID:Characterization of the insulin inhibition of the peptidolytic activities of the insulin-degrading enzyme-proteasome complex. 900 Jun 94

The mouse pancreatic beta TC3 and beta TC6-F7 cell lines were used to characterize the effects of interferon-gamma (IFN-y) on beta-cell phenotype and function. Initially, intracellular and secreted insulin were compared in glucose-stimulated cells over time. A significant reduction in insulin content and secretion was observed on a per-cell basis in glucose-stimulated beta TC3 and beta TC6-F7 cells after 12 h of exposure to IFN-gamma. The steadystate level of pre-proinsulin mRNA expression was not affected by IFN-gamma. Thus, we postulate that IFN-gamma's inhibitory actions occur after transcription of pre-proinsulin genes. Time-course analysis of IFN-gamma-regulated mRNA expression of the two intra-MHC-encoded subunits of the proteasome (low-molecular-mass polypeptide [Lmp]-2 and Lmp-7) revealed a correlation between their induction and the inhibitory effects of IFN-gamma on glucose-stimulated insulin production. Increased expression of Lmp-2 and Lmp-7 mRNA was accompanied by a corresponding induction of LMP2 and LMP7 protein expression. Subsequently, major histocompatibility complex (MHC) class I cell-surface expression was significantly increased in IFN-gamma-treated beta TC3 and beta TC6-F7 cells. Exposure of IFN-gamma-treated beta-cells to a peptide aldehyde inhibitor of the proteasome (MG132) significantly attenuated MHC class I cell-surface expression but did not prevent the negative effects of IFN-gamma on glucose responsiveness. Enhanced expression of the MHC class I antigen processing and presentation pathway and diminished insulin production appear to be distinct pathological alterations in beta-cells exposed to the insulitic cytokine IFN-gamma.
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PMID:Interferon-gamma independently activates the MHC class I antigen processing pathway and diminishes glucose responsiveness in pancreatic beta-cell lines. 913 43

Cellular homeostasis requires regulation of protein turnover. Protein degradation is an essential component of this process and is inhibited by insulin. The importance of cytosolic proteolysis in overall cellular protein degradation is increasingly apparent and an insulin effect on this system has been suggested but not proven. The present study shows that a membrane permeable substrate of the proteasome is degraded in HepG2 cells and that insulin inhibits its degradation both by isolated proteasomes and by intact cells. Inhibitors of the proteasome suppress degradation, and in the presence of these inhibitors insulin has no further effect. This is the first demonstration that insulin inhibition of cellular protein degradation is due to an effect on proteasomes.
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PMID:Insulin inhibition of proteasome activity in intact cells. 917 73

Autoimmune thyroid diseases (AITD) and insulin-dependent diabetes mellitus (IDDM) are two autoimmune syndromes of unknown etiology with common immune features. One is that the target cells, thyrocytes and pancreatic islet beta cells respectively, hyperexpress several proteins encoded in the HLA region: HLA class I, HLA class II and transporter associated with antigen processing (TAP-1): the clinical course and many aspects of the immunopathology are, however, quite different. Low-molecular-mass polypeptides 2 and 7 (LMP2 and LMP7) are proteasome subunits that increase the efficiency of endogenous antigen processing and are encoded in close vicinity to the TAP genes. We investigated whether LMP2 and LMP7 are hyperexpressed in thyrocytes and islet cells in AITD and IDDM. Thyroid tissue from Graves' disease patients (GD, n = 8) and Hashimoto thyroiditis (HT, n = 1) and pancreatic tissue from IDDM patients (n = 4) as well as control tissues were examined by the two-color indirect immunofluorescence technique. The results demonstrate that, in normal glands, thyrocytes and pancreatic islet cells express comparable moderate to low levels of LMP2 and LMP7. In AITD and IDDM, expression of LMP2/7 in the endocrine cells was disparate: while in AITD glands there was hyperexpression of LMP2 and 7 parallel to that of HLA class I and TAP-1, in the islet cells of recent onset diabetic pancreases (n = 2) the level of LMP2 and 7 expression was totally normal, including islets that were infiltrated by lymphocytes and hyperexpressed HLA class I and TAP-1. These observations suggest different mechanisms of endogenous peptides generation at the target cells in AITD from IDDM. Since this is a key step for the maintenance of peripheral tolerance, it may help to understand some of the different clinical features of the two autoimmune diseases.
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PMID:Proteasome subunits, low-molecular-mass polypeptides 2 and 7 are hyperexpressed by target cells in autoimmune thyroid disease but not in insulin-dependent diabetes mellitus: implications for autoimmunity. 927 25

Prior studies have shown that Madin-Darby canine kidney cells (MDCK) overexpressing the human insulin receptor bind and respond normally to insulin (T.C. Yeh, R.A. Roth, Diabetes 43 (1994) 1297-1303). Moreover, the insulin receptor preferentially localizes to the basolateral membrane of these cells. In the present studies, insulin was added to either the apical or the basolateral side of these cells and the extent of degradation of the insulin was assessed. Radioactive insulin added to either side was bound to its receptor and the radioactivity which reached the other side of the cell was to a large extent degraded fragments. Insulin added to the apical side was degraded to a larger extent (83%) than when added to the basolateral side (49%) although the basolateral side has much more insulin receptors than the apical side. This degradation process was not inhibitors of either lysosomal enzymes, the proteasome complex or cathepsins. The degradation process could however, be potently inhibited by the sulfhydryl alkylating agent N-ethylmaleimide. Further, cell surface biotinylation study showed that the insulin degrading enzyme was preferentially localized on the apical membranes. These results suggest that insulin added on the apical side of MDCK cells are more closely linked to the degradation process than that added on the basolateral side.
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PMID:Insulin degradation by Madin-Darby canine kidney cells expressing the insulin receptor. 927 78

HslVU in Escherichia coli a new two-component ATP-dependent protease composed of two heat-shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome beta-type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B-chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250-kDa complex) to HslU (a 450-kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20-fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2-3-fold in the presence of HslV. Concentrations of insulin B-chain that maximally stimulated the ATPase allowed maximal rates of the B-chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N-ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein-activated ATPase as well as an ATP-dependent proteinase, and these processes appear linked. Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP-dependent hydrolysis of peptides and proteins appear different.
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PMID:The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-dependent proteinase. 928 41


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