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 objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.
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PMID:Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B. 862 34

Lipopolysaccharide (LPS) stimulates the induction of the inducible isoform of nitric oxide synthase (iNOS) in part by inducing the nuclear translocation of the transcription factor nuclear factor-kappa B (NF-kappaB). LPS induces ubiquination and phosphorylation of the IkappaB inhibitory subunit of NF-kappaB. Subsequently, the ubiquitin-proteasome multicatalytic enzyme complex catalyzes the proteolytic degradation of IkappaB with resultant nuclear translocation of NF-kappaB. Our results demonstrate that the proteasome inhibitor calpain inhibitor I dose-dependently inhibited LPS-induced nitric oxide synthesis in RAW macrophages. The inhibitor was found to block iNOS transcription and protein translation as noted by Northern analysis and Western blotting, respectively. LPS stimulated rapid proteolytic degradation of IkappaB-alpha which was inhibited by approximately 50% in the presence of calpain inhibitor I. In contrast, LPS induced the delayed proteolytic degradation of IkappaB-beta which was almost totally inhibited by calpain inhibitor I. Calpain inhibitor I also decreased the LPS-induced nuclear translocation of NF-kappaB. These results demonstrate that the ubiquitin-proteasome complex has an important role in induction of iNOS in response to stimuli which act via the NF-kappaB/IkappaB signal transduction pathway. Furthermore, the results suggest that the ubiquitin-proteasome complex is important in the degradation of IkappaB-beta as well as IkappaB-alpha. Finally, we have demonstrated that there is a marked difference in the extent of proteolysis of IkappaB-alpha and IkappaB-beta when the ubiquitin-proteasome complex is inhibited with calpain inhibitor I.
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PMID:Inhibition of IkappaB-alpha and IkappaB-beta proteolysis by calpain inhibitor I blocks nitric oxide synthesis. 891 37

We report that exposure of aconitase to moderate concentrations of peroxynitrite, 3-morpholinosydnonimine (SIN-1; a superoxide- and nitric oxide-liberating substance), or hydrogen peroxide, inhibits the enzyme and enhances susceptibility to proteolytic digestion by the isolated 20 S proteasome. Exposure to more severe levels of oxidative stress, from these same agents, causes further inhibition of the enzymatic activity of aconitase but actually decreases its proteolytic breakdown by proteasome. It should be noted that the superoxide and nitric oxide liberated by SIN-1 decomposition react to form a steady flux of peroxynitrite. S-Nitroso-N-acetylpenicillamine, a compound that liberates nitric oxide alone, causes only a small loss of aconitase activity (25% or less) and has no effect on the proteolytic susceptibility of the enzyme. Proteasome also seems to be the main protease in cell lysates that can degrade aconitase after it has been oxidatively modified by exposure to peroxynitrite, SIN-1, or hydrogen peroxide. Using cell lysates isolated from K562 cells treated for several days with an antisense oligodeoxynucleotide to the initiation codon region of the C2 subunit of proteasome (a treatment which diminishes proteasome activity by 50-60%), the enhanced degradation of moderately damaged aconitase was essentially abolished. Other model proteins as well as complex mixtures of proteins, such as cell lysates, also exhibit enhanced proteolytic susceptibility after moderate SIN-1 treatment. Therefore we conclude that peroxynitrite reacts readily with proteins and that mild modification by peroxynitrite results in selective recognition and degradation by proteasome.
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PMID:Peroxynitrite increases the degradation of aconitase and other cellular proteins by proteasome. 955 59

Interleukin-1beta (IL-1beta) has been implicated as an effector molecule of beta-cell destruction in autoimmune diabetes. IL-1beta inhibits insulin secretion from pancreatic beta-cells by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. IL-1beta also induces the coexpression of the inducible isoform of cyclooxygenase (COX-2) that results in the overproduction of proinflammatory prostaglandins. The current studies were designed to characterize the involvement of protease(s) in the signaling pathway of IL-1beta-induced iNOS and COX-2 expression by rat islets and transformed rat pancreatic beta-cells. Because of the limitations of cell numbers of purified primary beta-cells obtained from rat islets, biochemical and molecular studies were performed using the rat insulinoma beta-cell line RINm5F. A serine protease inhibitor, Nalpha-P-tosyl-L-lysine chloromethyl ketone (TLCK), and a proteasome complex (26S) inhibitor, MG 132, inhibited IL-1beta-induced nitrite formation, an oxidation product of nitric oxide produced by iNOS, in a concentration-dependent manner, with complete inhibition observed at 100 micromol/l and 10 micromol/l, respectively. Both TLCK and MG 132 also inhibited iNOS gene expression at the level of mRNA and protein. In an analogous manner, TLCK (100 micromol/l) and MG 132 (10 micromol/l) inhibited IL-1beta-induced COX-2 enzyme activity (PGE2 formation) and COX-2 gene expression at the level of mRNA and protein. In human islets, the proteasome inhibitor MG 132 also inhibited the formation of the products of iNOS and COX-2 enzyme activity, nitrite, and PGE2, respectively. These findings suggest that the inhibitory action of TLCK and MG 132 on iNOS and COX-2 expression precedes transcription. The transcription factor NFkappaB is essential for activation of a number of cytokine-inducible enzymes and was evaluated as a possible site of protease action necessary for IL-1beta-induced coexpression of iNOS and COX-2. TLCK and MG 132 inhibited both IL-1beta-induced activation of NFkappaB and degradation of IkappaBalpha by islets and RINm5F cells. These results implicate protease activation as an early signaling event in IL-1beta-induced inhibition of beta-cell function. This study also suggests that IL-1beta-induced iNOS and COX-2 coexpression by pancreatic beta-cells share a common signaling pathway in utilizing the proteasome complex (26S) and the transcription factor NFkappaB, and it identifies sites of intervention to prevent the overproduction of their inflammatory products.
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PMID:Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells. 956 91

Myogenesis is characterized by membrane fusion and accumulation of muscle specific proteins. We have previously shown that nitric oxide acts as a messenger for membrane fusion. Here we show that inhibitors of the proteasome, such as lactacystin, reversibly block both the fusion of L6 myoblasts and the accumulation of muscle specific proteins, such as myosin heavy chain (MHC). The inhibitors also reversibly prevented the induction of the NF-kappaB activity, which is required for the expression of nitric oxide synthase (NOS). Moreover, the inhibition of the NF-kappaB activity occurred in parallel with that of the NOS activity upon treatment with increasing concentrations of lactacystin. While pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB, blocked both membrane fusion and accumulation of MHC, N(G)-monomethyl-L-arginine, a specific inhibitor of NOS, inhibited only the fusion. These results suggest that the proteasome plays an essential role in the regulation of myogenic differentiation through the activation of NF-kappaB and that the target of NF-kappaB for the expression of muscle specific proteins is distinct from that for myoblast fusion.
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PMID:Inhibitors of the proteasome block the myogenic differentiation of rat L6 myoblasts. 973 31

A growing number of physiologically relevant genes are regulated in response to changes in intracellular oxygen tension. It is likely that cells from a wide variety of tissues share a common mechanism of oxygen sensing and signal transduction leading to the activation of the transcription factor hypoxia-inducible factor 1 (HIF-1). Besides hypoxia, transition metals (Co2+, Ni2+ and Mn2+) and iron chelation also promote activation of HIF-1. Induction of HIF-1 by hypoxia is blocked by the heme ligands carbon monoxide and nitric oxide. There is growing, albeit indirect, evidence that the oxygen sensor is a flavoheme protein and that the signal transduction pathway involves changes in the level of intracellular reactive oxygen intermediates. The activation of HIF-1 by hypoxia depends upon signaling-dependent rescue of its alpha-subunit from oxygen-dependent degradation in the proteasome, allowing it to form a heterodimer with HIF-1beta (ARNT), which then translocates to the nucleus and impacts on the transcription of genes whose cis-acting elements contain cognate hypoxia response elements.
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PMID:Oxygen sensing and signaling: impact on the regulation of physiologically important genes. 1038 37

The ubiquitin/proteasome pathway mediates the degradation of many short-lived proteins that are critically involved in the regulation of cell proliferation and cell death, including the tumor suppressor protein p53. Accumulation of p53 and induction of apoptosis in RAW 264.7 macrophages in response to nitric oxide are well established. However, the molecular mechanisms involved in nitric oxide-induced p53 accumulation are unknown. Here we show that, similar to nitric oxide, treatment of macrophages with specific proteasome inhibitors, including clastolactacystin-beta-lactone, induces p53 accumulation and apoptosis, suggesting that nitric oxide may affect the activity of the proteasome. In support of this hypothesis, both exposure of cells to S-nitrosoglutathione and stimulation of endogenous nitric oxide production by lipopolysaccharide/interferon-gamma treatment result in inhibition of proteasome activity as measured in vitro by the degradation of the proteasome-specific substrate succinyl-Leu-Leu-Val-Tyr-4-methylcoumarin-7-amide. Moreover, chemically diverse nitric oxide donors interfere with proteasome-mediated degradation of polyubiquitinated p53 in vitro. These data imply that nitric oxide-induced apoptosis and accumulation of p53 are, at least in part, mediated by inhibition of the proteasome.
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PMID:Activation of the cell death program by nitric oxide involves inhibition of the proteasome. 1039 92

Cellular iron storage and uptake are coordinately regulated post-transcriptionally by cytoplasmic factors, iron-regulatory proteins 1 and 2 (IRP-1 and IRP-2). When iron in the intracellular transit pool is scarce, IRPs bind to iron-responsive elements (IREs) in the 5'-untranslated region of the ferritin mRNA and 3'-untranslated region of the transferrin receptor (TfR) mRNA. Such binding inhibits translation of ferritin mRNA and stabilizes the mRNA for TfR, whereas the opposite scenario develops when iron in the transit pool is plentiful. However, we (Richardson, D. R., Neumannova, V., Nagy, E., and Ponka, P. (1995) Blood 86, 3211-3219) and others reported that the binding of IRPs to IREs can also be modulated by nitric oxide (NO). In this study, we showed that a short exposure of RAW 264.7 cells (a murine macrophage cell line) to the NO(+) donor, sodium nitroprusside (SNP), caused a significant decrease in IRP-2 binding to the IREs followed by IRP-2 degradation and that these changes occurred without affecting IRP-1 binding. The SNP-mediated degradation of IRP-2 in RAW 264.7 cells could be prevented by MG-132 or lactacystin, known inhibitors of proteasome-dependent protein degradation. A SNP-mediated decrease in IRP-2 binding and levels was associated with a dramatic decrease in TfR mRNA levels and an increase in ferritin synthesis. Importantly, the proteasome inhibitor MG-132 prevented the SNP-mediated decrease in TfR mRNA levels. These observations suggest that IRP-2 can play an important role in controlling transferrin receptor expression.
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PMID:Control of transferrin receptor expression via nitric oxide-mediated modulation of iron-regulatory protein 2. 1055 72

Granuloma formation in response to mycobacterial infections is associated with increased expression of inducible nitric oxide synthase (NOS2) within granuloma macrophages and increased levels of nitrate/nitrite in the sera of infected mice. Continuous treatment with 5 mm or 10 mm l-N6-(1-imino-ethyl)-lysine (L-NIL), a selective NOS2-inhibitor, in acidified drinking water for up to 7 weeks consistently reduced infection-induced nitrate/nitrite to background levels in mycobacteria-infected BALB/c mice. Oral treatment with 5 mm L-NIL initiated at the time of infection significantly exacerbated growth of Mycobacterium tuberculosis, but had no effect on Mycobacterium avium colony-forming unit development in the liver, spleen and lungs of intravenously infected mice. In order to examine the role of nitric oxide in mycobacteria-induced granulomatous inflammation in the absence of any effect on the bacterial load, M. avium-infected mice were treated with 5 mm L-NIL from day 1 through 38 and the development of granulomatous lesions in the liver was assessed by histology, immunohistology and reverse-transcription-polymerase chain reaction (RT-PCR). Computer- and video-assisted morphometry performed at 4 and 7 weeks post-infection showed that treatment with L-NIL led to markedly increased number, cellularity and size of granulomatous lesions in infected mice regardless of the virulence of the M. avium isolate used for infection. Immunohistology of the liver revealed that in mice treated with L-NIL, the numbers of CD3+ T cells, CD21/35+ B cells, CD11b+ macrophages and RB6-8C5+ granulocytes associated with granulomatous lesions was increased. RT-PCR of the liver showed that in L-NIL-treated mice infected with M. avium, mRNA levels of tumour necrosis factor, interleukin-12p40, interferon-gamma, interleukin-10 and interferon-gamma-inducible protein-10 (IP-10) were up-regulated, while mRNA levels of interleukin-4, monocyte chemotactic protein-1 (MCP-1) and MCP-5 were similar to those in untreated control infected mice. When M. avium-infected mice were treated with 5 mm L-NIL between the 5th and 12th weeks of infection, similar changes in granuloma number and size were found in the absence of any effect on the bacterial load. These findings demonstrate that nitric oxide regulates the number, size and cellular composition of M. avium-induced granulomas independently of antibacterial effects by modulating the cytokine profile within infected tissues.
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PMID:NOS2-derived nitric oxide regulates the size, quantity and quality of granuloma formation in Mycobacterium avium-infected mice without affecting bacterial loads. 1058 88

Recipient IgG immunity against leukoreduced donor platelets is dependent on indirect T-cell allorecognition and is suppressed in vivo by inhibitors (aminoguanidine, AMG) of inducible nitric oxide synthase (iNOS). To examine recipient processing pathways of donor platelet antigens, enriched macrophages (antigen-presenting cells [APC]) from BALB/c (H-2(d)) mice were pulsed with allogeneic C57BL/6 (H-2(b)) platelets and transfused weekly into naive BALB/c mice. Platelet-pulsed APC stimulated IgG antidonor antibody production in 45% of recipients by the second transfusion and in 100% by the sixth transfusion; this response was enhanced by pulsing in the presence of interferon-gamma. By the sixth transfusion, high-titer IgG1 (mean titer 4990) and IgG2a (1933) isotypes specific for donor major histocompatibility complex (MHC) class I antigens were detected. Platelet pulsing in the presence of AMG or colchicine significantly inhibited the ability of APC to stimulate IgG alloantibodies; only 50% (P <.005) and 20% (P <.0001) of recipients, respectively, produced antibodies by the sixth transfusion. AMG inhibition was reversed by the addition of L-arginine, the substrate for iNOS. In contrast, pulsing in the presence of chloroquine, the proteasome inhibitory peptide MG115, or Brefeldin A enhanced APC immunity (70-100% of recipients antibody positive by the second transfusion [P <.05]); these agents allowed the pulsed APC to stimulate IgG2a but inhibited IgG1 production and this correlated with a reduction in serum interleukin (IL)-4 levels. The results suggest that for donor platelet antigens to stimulate IgG alloantibodies, recipient APC use the essential generation of nitric oxide and a noncytosolic, pH-independent processing pathway, which can be exploited as an effective immunotherapy target to further inhibit alloimmunization against leukoreduced platelets. (Blood. 2000;95:1735-1742)
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PMID:Unique processing pathways within recipient antigen-presenting cells determine IgG immunity against donor platelet MHC antigens. 1068 32


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