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)

The aim of the present study was to characterize human CYP2E1 turnover and examine the possible proteolytic pathways responsible for the rapid degradation of CYP2E1 in a transfected HepG2 cell line expressing human CYP2E1. Two methods were used to study the CYP2E1 turnover; after addition of cycloheximide, the half-life of the CYP2E1 in the intact cells was about 6 h as detected by PNP catalytic activity assay and immunoblot analysis of apoprotein content. CYP2E1 substrates or ligands such as 4-methylpyrazole, ethanol, glycerol, and dimethyl sulfoxide protected CYP2E1 against this rapid degradation, whereas CCl4 accelerated this process. The second procedure involved pulse-chase experiments after labeling CYP2E1 with [35S]methionine and immunoprecipitation with anti-human CYP2E1 IgG. The half-life of CYP2E1 was about 2.5 h, and the various substrates or ligands modified the turnover process within intact cells as described for the cycloheximide experiments. More than 20 different reagents including antioxidants, physiological metabolites, lysosomal inhibitors, and protease inhibitors were screened for possible effects on CYP2E1 proteolytic degradation. Dibutyryl cAMP had no effect on CYP2E1 activity or turnover. Among those reagents tested so far, the serine protease inhibitor 1-chloro-3-tosylamido-7-amino-2-heptanone hydrochloride exhibited some protection against CYP2E1 degradation. To demonstrate whether the proteasome complex is involved in this process, Czb-Ile-Glu(OtBu)-Ala-leucinal (PSI) as a cell penetrating aldehydic proteasome inhibitor and Czb-Leu-norleucinal (calpeptin inhibitor) as an aldehydic nonproteosomal protease inhibitor were used to examine their effect on both the normal and the CCl4-stimulated CYP2E1 proteolytic degradation pathways. Treatment with PSI at concentrations ranging from 5 to 80 microM resulted in a dose-dependent protection against the loss of both the normal CYP2E1 and the CCl4-modified CYP2E1. The maximum protection by PSI at a concentration of 80 microM after a 12-h chase period was about 60% in cells treated with 2 mM CCl4 or 75% in cells without CCl4 treatment. Calpeptin inhibitor afforded little or no protection against CYP2E1 degradation in the absence or presence of CCl4. PSI did not inhibit CYP2E1 catalytic activity, suggesting that it was not a ligand for CYP2E1. These results indicate that human CYP2E1 has a short half-life span and that substrates can significantly modify its turnover rate in intact HepG2 cells. The proteasome proteolytic pathway may be involved in the degradation process of both the normal and the CCl4-modified human CYP2E1 in this model.
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PMID:Characterization of cytochrome P4502E1 turnover in transfected HepG2 cells expressing human CYP2E1. 914 49

In chronic renal failure (CRF), the ATP-dependent, ubiquitin-proteasome proteolytic pathway is activated with concurrent increases in the transcription of genes encoding proteins of this pathway in muscle. We have shown that the stimuli for these responses include acidosis and glucocorticoids, but other endocrine abnormalities in CRF (e.g., insulin resistance) could contribute to these responses. In fact, a major effect of insulin in muscle is to suppress protein degradation. To examine whether insulin influences the ubiquitin-proteasome pathway, we measured protein degradation in incubated epitrochlearis muscles of diabetic and pair-fed control rats. Muscle proteolysis was increased in pathways that do not involve lysosomes or Ca(2+)-dependent proteases; but MG132, a protease inhibitor that blocks ATP synthesis, eliminated the accelerated rate of protein degradation in diabetic rat muscles. Diabetes mellitus also increased levels of mRNAs encoding ubiquitin (334%), E2 ubiquitin-conjugating enzyme (247%), and the C3 (320%), C5 (349%), and C9 (216%) proteasome subunits in muscle. Finally, transcription of the ubiquitin gene in diabetic rat muscles was increased. Diabetic rats were acidotic, but eliminating acidemia by giving NaHCO3 did not block the increase in muscle proteolysis. Giving diabetic rats insulin prevented the excessive muscle proteolysis, suggesting that insulin acts as a suppressor of the ubiquitin-proteasome pathway. Thus, the insulin resistance of uremia could contribute to muscle protein wasting in CRF.
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PMID:Signals regulating accelerated muscle protein catabolism in uremia. 938 16

Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1beta induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1beta induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1beta-induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-kappaB-mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-kappaB translocation to the nucleus, norLeu had no effect on NF-kappaB nuclear translocation or IL-1beta-induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1beta-induced MCP-1 gene expression in human endothelial cells.
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PMID:IL-1beta-induced monocyte chemoattractant protein-1 gene expression in endothelial cells is blocked by proteasome inhibitors. 963 34

Pseudomonas fluorescens, a gram-negative psychrotrophic bacterium, secretes a thermostable lipase into the extracellular medium. In our previous study, the lipase of P. fluorescens SIK W1 was cloned and expressed in Escherichia coli, but it accumulated as inactive inclusion bodies. Amino acid sequence analysis of the lipase revealed a potential C-terminal targeting sequence recognized by the ATP-binding cassette (ABC) transporter. The genetic loci around the lipase gene were searched, and a secretory gene was identified. Nucleotide sequencing of an 8.5-kb DNA fragment revealed three components of the ABC transporter, tliD, tliE, and tliF, upstream of the lipase gene, tliA. In addition, genes encoding a protease and a protease inhibitor were located upstream of tliDEF. tliDEF showed high similarity to ABC transporters of Pseudomonas aeruginosa alkaline protease, Erwinia chrysanthemi protease, Serratia marcescens lipase, and Pseudomonas fluorescens CY091 protease. tliDEF and the lipase structural gene in a single operon were sufficient for E. coli cells to secrete the lipase. In addition, E. coli harboring the lipase gene secreted the lipase by complementation of tliDEF in a different plasmid. The ABC transporter of P. fluorescens was optimally functional at 20 and 25 degrees C, while the ABC transporter, aprD, aprE, and aprF, of P. aeruginosa secreted the lipase irrespective of temperature between 20 and 37 degrees C. These results demonstrated that the lipase is secreted by the P. fluorescens SIK W1 ABC transporter, which is organized as an operon with tliA, and that its secretory function is temperature dependent.
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PMID:Identification of the tliDEF ABC transporter specific for lipase in Pseudomonas fluorescens SIK W1. 1007 78

Withdrawal of trophic support from growth factor-dependent MO7e human myeloid progenitor cells induces apoptosis characterized by DNA fragmentation and degradation of the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Inhibitors of caspase (ICE) protease family members did not inhibit apoptosis or DNA fragmentation induced by factor withdrawal, but blocked degradation of DNA-PKcs. Thus, caspase activity accounts for only a component of the apoptotic program in MO7e hematopoietic cells. The protease inhibitor TPCK, but not other protease inhibitors, blocked DNA fragmentation, but not degradation of DNA-PKcs during apoptosis of MO7e cells. Thus, caspase-independent and caspase-dependent protease cascades mediate distinct features of MO7e cell apoptosis. The proteasome inhibitors calpain inhibitor I and lactacystin promoted DNA fragmentation, degradation of DNA-PKcs and apoptosis of MO7e cells. The ability of lactacystin to promote DNA fragmentation was abrogated by TPCK, but not by caspase inhibitors, whereas the ability of lactacystin to promote degradation of DNA-PKcs was blocked by caspase inhibitors, but not by TPCK. Thus, caspase-dependent and caspase-independent protease cascades are downstream of and regulated by the proteasome, which plays a central role in regulating the multiple protease cascades that induce apoptosis.
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PMID:The proteasome regulates caspase-dependent and caspase-independent protease cascades during apoptosis of MO7e hematopoietic progenitor cells. 1034 10

p107 protein, a member of the retinoblastoma family protein, suppresses growth promotion in cancer cells. We have already reported evidence that calpain, a calcium dependent protease is involved in the cleavage of p107 protein. We show here that p107 protein can also be a substrate for ubiquitination. A negative growth regulator, the HMG-CoA reductase inhibitor lovastatin was found to induce loss of p107 protein which was reversible by a specific protease inhibitor lactacystin as well as calpain inhibitor. Following treatment with lovastatin higher molecular weight ubiquitinated forms of p107 were detected by anti-p107 immunoprecipitation and anti-ubiquitin Western blotting. These forms further increased when lactacystin was added to culture medium. These results indicate that ubiquitin-proteasome pathway plays a potential role in the degradation as well as calpain. The data presented here suggest a model in which calpain and ubiquitin-proteasome system possibly play a cooperative role in targeting the protein under certain conditions.
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PMID:Proteolytic degradation of the retinoblastoma family protein p107: A putative cooperative role of calpain and proteasome. 1053 70

The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years. Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease. In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM). In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced. In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome. Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir. At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase. Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded. Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.
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PMID:How an inhibitor of the HIV-I protease modulates proteasome activity. 1058 54

Guanabenz, a metabolism-based irreversible inactivator of neuronal nitric-oxide synthase (nNOS) in vitro, causes the loss of immunodetectable nNOS in vivo. This process is selective in that the slowly reversible inhibitor N(G)-nitro-L-arginine did not decrease the levels of nNOS in vivo. To better understand the mechanism for the loss of nNOS protein in vivo, we have investigated the effects of guanabenz and N(G)-nitro-L-arginine in HEK 293 cells stably transfected with the enzyme. We show here that guanabenz, but not N(G)-nitro-L-arginine, caused the inactivation and loss of nNOS protein in the HEK 293 cells. In studies with cycloheximide or in pulse-chase experiments with [(35)S]methionine, we demonstrate that the loss of nNOS was due in large part to enhanced proteolysis of the protein with the half-life decreasing by one-half from 20 to 10 h. Other metabolism-based irreversible inactivators to nNOS, N(G)-methyl-L-arginine, and N(5)-(1-iminoethyl)-L-ornithine, but not the reversible inhibitor 7-nitroindazole (7-NI), caused a similar decrease in the half-life of nNOS. Proteasomal inhibitors, lactacystin, Cbz-leucine-leucine-leucinal, and N-acetyl-leucine-leucine-norleucinal, but not the lysosomal protease inhibitor leupeptin, were found to effectively inhibit the proteolytic degradation of nNOS. Thus we have shown for the first time that the irreversible inactivators of nNOS, perhaps through covalent alteration of the enzyme, enhance the proteolytic turnover of the enzyme by a mechanism involving the proteasome.
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PMID:Guanabenz-mediated inactivation and enhanced proteolytic degradation of neuronal nitric-oxide synthase. 1064 88

A novel trypsin-type serine proteinase, which processes the precursors of the envelope fusion glycoproteins of pneumotropic Sendai and human influenza A viruses, was purified to homogeneity from pig lungs. On SDS/PAGE, the purified enzyme gave a protein band corresponding to about 32 kDa, and has an apparent molecular mass of 120 kDa, as determined by gel permeation chromatography. Immunohistochemical staining with antibodies against this enzyme revealed that the enzyme is located in pig lung mast cells. The N-terminal 44-amino-acid sequence of the enzyme exhibits about 80% identity with those of mast cell tryptases from other species. Of the inhibitors tested, di-isopropyl fluorophosphate, antipain, leupeptin, benzamidine and a few proteinaceous inhibitors, such as mucus protease inhibitor and aprotinin, inhibited this enzyme activity. Heparin stabilized the enzyme, but high-ionic-strength conditions did not, unlike for human mast cell tryptase. The purified enzyme efficiently processed the fusion glycoprotein precursor of Sendai virus and slowly processed hemagglutinin of human influenza A virus, and triggered the infectivity of Sendai virus in a dose-dependent manner, although human mast cell tryptase beta and rat mast cell tryptase (rat MCP-7) from lungs did not process these fusion glycoproteins at all. These results suggest that mast cell tryptase in pig lungs is the possible trigger of the pneumotropic virus infections.
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PMID:Mast cell tryptase from pig lungs triggers infection by pneumotropic Sendai and influenza A viruses. Purification and characterization. 1082 3

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. We demonstrated that treatment of THP-1 human monocytic leukemia cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death through an apoptotic pathway. Apoptosis in THP-1 cells induced by Z-LLL-CHO involved a cytochrome c-dependent pathway, which included the release of mitochondrial cytochrome c, activation of caspase-9 and -3, and cleavage of Bcl-2 into a shortened 22-kDa fragment. Induction of apoptosis by protease inhibitor also was detected in U937 and TF-1 leukemia cell lines and cells obtained from acute myelogenous leukemia patients but not in normal human blood monocytes. Treatment of human blood monocytes with Z-LLL-CHO did not induce apoptosis or Bcl-2 cleavage in these cells that rarely proliferate. Interestingly, when THP-1 cells were induced to undergo monocytic differentiation by bryostatin 1, a naturally occurring protein kinase C activator, they were no longer susceptible to apoptosis induced by Z-LLL-CHO. Bryostatin 1-induced differentiation of THP-1 cells was associated with growth arrest, acquisition of adherent capacity, and expression of membrane markers characteristic of blood monocytes. Likewise, differentiated THP-1 cells were refractory to Z-LLL-CHO-induced cytochrome c release, caspase activation, and Bcl-2 cleavage. Resistance to Z-LLL-CHO-induced apoptosis in differentiated THP-1 cells was not due to cell cycle arrest. These findings show that the action of proteasome inhibitors is mediated primarily through a cytochrome c-dependent pathway and induces apoptosis in leukemic cells that are not differentiated.
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PMID:Human THP-1 monocytic leukemic cells induced to undergo monocytic differentiation by bryostatin 1 are refractory to proteasome inhibitor-induced apoptosis. 1096 81


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