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 transcription factor NF-kappa-B is normally sequestered in the cytoplasm by its inhibitory subunit IkappaB. Most extracellular signals activate NF-kappa-B through a mechanism involving the phosphorylation and proteasome-dependent degradation of IkappaB. EGF activates NF-kappaB in A-431 carcinoma cells, which overexpress EGF receptors and in mouse embryo fibroblasts, which have a normal complement of receptors. Supershift experiments indicate that the NF-kappa-B complexes induced by EGF are composed of p50/p50 homodimers and p65/p50 heterodimers, but not c-rel. EGF stimulation enhances the degradation of IkappaBalpha, but not IkappaBbeta nor an N-terminal deletion mutant of IkappaBalpha. Treatment of cells with a proteasome inhibitor, such as ALLN or MG132, blocks EGF-mediated NF-kappaB activation, indicating that EGF-induced NF-kappa-B activation requires proteasome-dependent IkappaB degradation. Also, Bapta A/M (a cell-permeable chelator of intracellular calcium) blocks EGF-induced NF-kappa-B activation and IkappaBalpha degradation, suggesting a requirement of intracellular free Ca2+ for this growth factor response. Protein kinase C inhibition, in contrast, did not influence EGF activation of NF-kappaB.
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PMID:Epidermal growth factor activation of NF-kappaB is mediated through IkappaBalpha degradation and intracellular free calcium. 957 90

Previously we showed that infection of human type II airway epithelial (A549) cells with purified respiratory syncytial virus (pRSV) induced interleukin-8 transcription by a mechanism involving cytokine-inducible cytoplasmic-nuclear translocation of the RelA transcription factor. In unstimulated cells, RelA is tethered in the cytoplasm by association with the IkappaB inhibitor and can be released only following IkappaB degradation. In this study, we examined the spectrum of IkappaB isoform expression and kinetics of proteolysis of the isoforms in A549 cells following pRSV infection. In contrast to the rapid and robust activation of RelA DNA binding that peaked within 15 min of treatment produced by the prototypic activator tumor necrosis factor alpha (TNF-alpha), pRSV produced a weaker increase in RelA binding that began at 3 h and did not peak until 24 h after infection. A549 cells expressed the IkappaB inhibitory subunits IkappaBalpha, IkappaBbeta, and p105; however, following either stimulus, only the IkappaBalpha and IkappaBbeta steady-state levels declined in parallel with the increase in RelA DNA-binding activity. The >120-min half-life of IkappaBalpha in control cells was shortened to 5 min in TNF-alpha-stimulated cells and to 90 min in pRSV-infected cells. Although IkappaBalpha was resynthesized within 30 min following recombinant human TNFalpha treatment due to a robust 25-fold increase of IkappaBalpha mRNA expression (the RelA:IkappaBalpha positive feedback loop), following pRSV infection, there was no reaccumulation of IkappaBalpha protein, as infected cells produced only a 3-fold increase in IkappaBalpha mRNA at 24 h, indicating the RelA:IkappaBalpha positive feedback loop was insufficient to restore control IkappaBalpha levels. IkappaBalpha proteolysis induced by TNF-alpha occurred through the 26S proteasome, as both 26S proteasome activity and IkappaBalpha proteolysis were blocked by specific inhibitors lactacystin, MG-132, and ZLLF-CHO. Although total proteasome activity in 24-h pRSV-infected lysates increased twofold, its activity was >90% inhibited by the proteasome inhibitors; surprisingly, however, IkappaBalpha proteolysis was not. We conclude that RSV infection produces IkappaBalpha proteolysis through a mechanism primarily independent of the proteasome pathway.
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PMID:The major component of IkappaBalpha proteolysis occurs independently of the proteasome pathway in respiratory syncytial virus-infected pulmonary epithelial cells. 957 51

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

Nuclear factor kappaB (NFkappaB) is a ubiquitously expressed transcription factor that is regulated by the cytoplasmic inhibitor protein IkappaBalpha. Biological agents such as tumor necrosis factor alpha (TNFalpha), which activate NFkappaB, result in the rapid degradation of IkappaBalpha. Adenoviral-mediated gene transfer of Bcl-2 prevents apoptosis of neonatal ventricular myocytes induced by TNFalpha. In view of the growing evidence that NFkappaB may play an important role in regulating apoptosis, we determined whether TNFalpha and Bcl-2 could modulate the activity of NFkappaB in ventricular myocytes. Stimulation of myocytes with TNFalpha resulted in a 2.1-fold increase (p < 0.001) in NFkappaB-dependent gene transcription and nuclear DNA binding. Similarly, a 1.9-fold increase (p < 0.0002) in NFkappaB-dependent gene transcription was observed in myocytes expressing Bcl-2. Nuclear DNA binding activity of NFkappaB was significantly increased in myocytes expressing Bcl-2, with a concomitant reduction in IkappaBalpha protein level. The Bcl-2-mediated loss of IkappaBalpha could be prevented by the proteasome inhibitor lactacystin, consistent with the notion that the targeted degradation of IkappaBalpha consequent to overexpression of Bcl-2 utilizes the ubiquitin-proteasome pathway. This was further tested in human 293 cells in which the N-terminal region of IkappaBalpha was identified to be an important regulatory site for Bcl-2. Deletion of this region or a serine to alanine substitution mutant at amino acids 32 and 36, which are defective for both phosphorylation and degradation, were more resistant than wild type IkappaBalpha to the inhibitory effects of Bcl-2. To our knowledge, this provides the first evidence for the regulation of IkappaBalpha by Bcl-2 and suggests a link between Bcl-2 and the NFkappaB signaling pathway in the suppression of apoptosis.
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PMID:Bcl-2 activates the transcription factor NFkappaB through the degradation of the cytoplasmic inhibitor IkappaBalpha. 972 9

Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. We have detected modified IkappaBalpha, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugated SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.
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PMID:SUMO-1 modification of IkappaBalpha inhibits NF-kappaB activation. 973 60

We examined the mechanisms by which two different types of photonic radiation, short wavelength UV (UV-C) and gamma radiation, activate transcription factor NF-kappaB. Exposure of mammalian cells to either form of radiation resulted in induction with similar kinetics of NF-kappaB DNA binding activity, nuclear translocation of its p65(RelA) subunit, and degradation of the major NF-kappaB inhibitor IkappaBalpha. In both cases, induction of NF-kappaB activity was attenuated by proteasome inhibitors and a mutation in ubiquitin-activating enzyme, suggesting that both UV-C and gamma radiation induce degradation of IkappaBs by means of the ubiquitin/proteasome pathway. However, although the induction of IkappaBalpha degradation by gamma rays was dependent on its phosphorylation at Ser-32 and Ser-36, UV-C-induced IkappaBalpha degradation was not dependent on phosphorylation of these residues. Even the "super repressor" IkappaBalpha mutant, which contains alanines at positions 32 and 36, was still susceptible to UV-C-induced degradation. Correspondingly, we found that gamma radiation led to activation of IKK, the protein kinase that phosphorylates IkappaBalpha at Ser-32 and Ser-36, whereas UV-C radiation did not. Furthermore, expression of a catalytically inactive IKKbeta mutant prevented NF-kappaB activation by gamma radiation, but not by UV-C. These results indicate that gamma radiation and UV-C activate NF-kappaB through two distinct mechanisms.
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PMID:Ionizing radiation and short wavelength UV activate NF-kappaB through two distinct mechanisms. 978 32

The transcription factor NF-kappaB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-kappaB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IkappaB is degraded by the ubiquitin-proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-kappaB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin-proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-kappaB and the subsequent transcription of genes that are regulated by NF-kappaB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IkappaBalpha degradation and NF-kappaB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin-proteasome pathway and NF-kappaB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.
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PMID:Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis. 986 Oct 28

The cytokine tumor necrosis factor alpha (TNF-alpha) induces expression of inflammatory gene networks by activating cytoplasmic to nuclear translocation of the nuclear factor-kappaB (NF-kappaB) transcription factor. NF-kappaB activation results from sequential phosphorylation and hydrolysis of the cytoplasmic inhibitor, IkappaBalpha, through the 26 S proteasome. Here, we show a parallel proteasome-independent pathway for cytokine-inducible IkappaBalpha proteolysis in HepG2 liver cells mediated by cytosolic calcium-activated neutral protease (calpains). Pretreatment with either calpain- or proteasome-selective inhibitors partially blocks up to 50% of TNF-alpha-inducible IkappaBalpha proteolysis; pretreatment with both is required to completely block IkappaBalpha proteolysis. Similarly, in transient cotransfection assays, expression of the specific inhibitor, calpastatin, partially blocks TNF-alpha-inducible NF-kappaB-dependent promoter activity and IkappaBalpha proteolysis. In TNF-alpha-stimulated cells, a rapid (within 1 min), 2.2-fold increase in cytosolic calpain proteolytic activity is measured using a specific fluorescent assay. Inducible calpain proteolytic activity occurs coincidentally with the particulate-to-cytosol redistribution of the catalytic m-calpain subunit into the IkappaBalpha compartment. Addition of catalytically active m-calpain into broken cells was sufficient to produce ligand-independent IkappaBalpha proteolysis and NF-kappaB translocation. As additional evidence for calpain-dependent IkappaBalpha proteolysis and NF-kappaB activation, we demonstrate that this process occurs in a cell line (ts20b) deficient in the ubiquitin-proteasome pathway. Following inactivation of the temperature-sensitive ubiquitin-activating enzyme, IkappaBalpha proteolysis occurs in a manner sensitive only to calpain inhibitors. Our results demonstrate that TNF-alpha activates cytosolic calpains, a parallel pathway that degrades IkappaBalpha and activates NF-kappaB activation independently of the ubiquitin-proteasome pathway.
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PMID:Tumor necrosis factor-alpha-inducible IkappaBalpha proteolysis mediated by cytosolic m-calpain. A mechanism parallel to the ubiquitin-proteasome pathway for nuclear factor-kappab activation. 987 17

The ATP/ubiquitin-dependent 26S proteasome is a central regulator of cell cycle progression and stress responses. While investigating the application of peptide aldehyde proteasome inhibitors to block signal-induced IkappaBalpha degradation in human LNCaP prostate carcinoma cells, we observed that persistent inhibition of proteasomal activity signals a potent cell death program. Biochemically, this program included substantial upregulation of PAR-4 (prostate apoptosis response-4), a putative pro-apoptotic effector protein and stabilization of c-jun protein, a potent pro-death effector in certain cells. We also observed modest downregulation of bcl-XL, a pro-survival effector protein. However, in contrast to some recent reports stable, high level, expression of functional bcl-2 protein in prostate carcinoma cells failed to signal protection against cell death induction by proteasome inhibitors. Also in disagreement to a recent report, no evidence was found for activation of the JNK stress kinase pathway. A role for p53, a protein regulated by the proteasome pathway, was ruled out, since comparable cell death induction by proteasome inhibitors occurred in PC-3 cells that do not express functional p53 protein. These data signify that the ubiquitin/proteasome pathway represents a potential therapeutic target for prostate cancers irrespective of bcl-2 expression or p53 mutations.
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PMID:Prostate carcinoma cell death resulting from inhibition of proteasome activity is independent of functional Bcl-2 and p53. 987 95

Nuclear factor kappa B (NF-kappaB) is an important transcription factor for the genes of many pro-inflammatory proteins and is strongly activated by the cytokines interleukin-1 and tumor necrosis factor (TNF)alpha under various pathological conditions. In nonstimulated cells, NF-kappaB is present in the cytosol where it is complexed to its inhibitor IkappaB. Activation of NF-kappaB depends on the signal-induced phosphorylation of IkappaB by specific IkappaB kinases which initiates the inhibitor's conjugation to ubiquitin and subsequent degradation by the proteasome. We used both TNF-stimulated and okadaic-acid-stimulated HeLa cells to purify three biochemically distinct kinase activities targeting one or both of the two serines (S32 and S36) in IkappaBalpha which induce its rapid degradation upon cytokine stimulation. All three activities correspond to known IkappaB kinases: the mitogen-activated 90 kDa ribosomal S6 kinase (p90rsk1), the IkappaB kinase 1/2 complex (IKK1/2) and casein kinase II (CK II). However, we found that only one of the activities, namely the IKK1/2 complex, exists as a pre-assembled kinase-substrate complex in which the IKKs are directly or indirectly associated with several NF-kappaB-related and IkappaB-related proteins: RelA, RelB, cRel, p100, p105, Ikappa Balpha, Ikappa Bbeta and Ikappa Bepsilon. The existence of stable kinase-substrate complexes, the presence of all three known IkappaB isoforms in these complexes and our observation that the IKK complex is capable of phosphorylating Ikappa Balpha-, Ikappa Bbeta- and Ikappa Bepsilon-derived peptides at the respective degradation-relevant serines suggests that the IKK complex exerts a broad regulatory role for the activation of different NF-kappaB species. In contrast to previous studies, which locate CK II phosphorylation sites exclusively to the C-terminal PEST sequence of Ikappa Balpha, we observed efficient phosphorylation of serine 32 in Ikappa Balpha by the purified endogenous CK II complex. Therefore, both p90rsk1 and CK II have the same preference for phosphorylating only one of the two serines which are relevant for inducible degradation.
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PMID:All three IkappaB isoforms and most Rel family members are stably associated with the IkappaB kinase 1/2 complex. 991


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