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)

Interaction of many infectious agents with eukaryotic host cells is known to cause activation of the ubiquitous transcription factor nuclear factor kappaB (NF-kappaB) (U. Siebenlist, G. Franzoso, and K. Brown, Annu. Rev. Cell Biol. 10:405-455, 1994). Recently, we reported a biphasic pattern of NF-kappaB activation in cultured human umbilical vein endothelial cells consequent to infection with Rickettsia rickettsii, an obligate intracellular gram-negative bacterium and the etiologic agent of Rocky Mountain spotted fever (L. A. Sporn, S. K. Sahni, N. B. Lerner, V. J. Marder, D. J. Silverman, L. C. Turpin, and A. L. Schwab, Infect. Immun. 65:2786-2791, 1997). In the present study, we describe activation of NF-kappaB in a cell-free system, accomplished by addition of partially purified R. rickettsii to endothelial cell cytoplasmic extracts. This activation was rapid, reaching maximal levels at 60 min, and was dependent on the number of R. rickettsii organisms added. Antibody supershift assays using monospecific antisera against NF-kappaB subunits (p50 and p65) confirmed the authenticity of the gel-shifted complexes and identified both p50-p50 homodimers and p50-p65 heterodimers as constituents of the activated NF-kappaB pool. Activation occurred independently of the presence of endothelial cell membranes and was not inhibited by removal of the endothelial cell proteasome. Lack of involvement of the proteasome was further confirmed in assays using the peptide-aldehyde proteasome inhibitor MG 132. Activation was not ATP dependent since no change in activation resulted from addition of an excess of the unhydrolyzable ATP analog ATPgammaS, supplementation with exogenous ATP, or hydrolysis of endogenous ATP with ATPase. Furthermore, Western blot analysis before and after in vitro activation failed to demonstrate phosphorylation of serine 32 or degradation of the cytoplasmic pool of IkappaB alpha. This lack of IkappaB alpha involvement was supported by the finding that R. rickettsii can induce NF-kappaB activation in cytoplasmic extracts prepared from T24 bladder carcinoma cells and human embryo fibroblasts stably transfected with a superrepressor phosphorylation mutant of IkappaB alpha, rendering NF-kappaB inactivatable by many known signals. Thus, evidence is provided for a potentially novel NF-kappaB activation pathway wherein R. rickettsii may interact with and activate host cell transcriptional machinery independently of the involvement of the proteasome or known signal transduction pathways.
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PMID:Proteasome-independent activation of nuclear factor kappaB in cytoplasmic extracts from human endothelial cells by Rickettsia rickettsii. 957 57

Increased gene expression as a consequence of environmental stress is typically observed in mammalian cells. In the past few years the cis- and trans-acting genetic elements responsible for gene induction by radiation (from UV-C to visible light) started to be well characterized. The molecular mechanisms involved in the cell response to radiation reveal that an important control occurs at the transcriptional level and is coordinated by various transcription factors. Among these transcription factors, the well-known Rel/NF-kappa B family of vertebrate transcription factors plays a pivotal role as it controls both the inflammatory and immune responses. The NF-kappa B family comprises a number of structurally related, interacting proteins that bind DNA as dimers and whose activity is regulated by subcellular location. This family includes many members (p50, p52, RelA, RelB, c-Rel, ...), most of which can form DNA-binding homo- or heterodimers. Nuclear expression and consequent biological action of the eukaryotic NF-kappa B transcription factor complex are tightly regulated through its cytoplasmic retention by ankyrin-rich inhibitory proteins known as I kappa B. In the best-characterized example, I kappa B-alpha interacts with a p50/RelA (NF-kappa B) heterodimer to retain the complex in the cytoplasm and inhibit its DNA-binding activity. Upon receiving a variety of signals, many of which are probably mediated by the generation of reactive oxygen species (ROS), I kappa B-alpha undergoes phosphorylation, is then ubiquitinated at nearby lysine residues and finally degraded by the proteasome, while still complexed with NF- kappa B. Removal of I kappa B-alpha uncovers the nuclear localization signals on subunits of NF-kappa B, allowing the complex to enter the nucleus, bind to DNA and affect gene expression. In this paper, we shall show that molecular mechanisms leading to NF-kappa B activation by UV or by photosensitization are initiated by oxidative damage at the membrane level or by the induction of DNA alterations. While the exact nature of the transduction intermediates is still unknown, we shall show that NF-kappa B activation by radiation follows different pathways from those used by pro-inflammatory cytokines.
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PMID:Nf-kappa B: an important transcription factor in photobiology. 981 95

Neurotrophins activate multiple signaling pathways in neurons. However, the precise roles of these signaling molecules in cell survival are not well understood. In this report, we show that nerve growth factor (NGF) activates the transcription factors NF-kappaB and AP-1 in cultured sympathetic neurons. Activated NF-kappaB complexes were shown to consist of heterodimers of p50 and Rel proteins (RelA, as well as c-Rel), and NF-kappaB activation was found to occur independently of de novo protein synthesis but in a manner that required the action of the proteasome complex. Treatment with the NF-kappaB inhibitory peptide SN50 in the continuous presence of NGF resulted in dose-dependent induction of cell death. Under the conditions used, SN50 was shown to selectively inhibit NF-kappaB activation but not the activation of other cellular transcription factors such as AP-1 and cAMP response element-binding protein. Cells treated with SN50 exhibited morphological and biochemical hallmarks of apoptosis, and the kinetics of cell killing were accelerated relative to death induced by NGF withdrawal. Finally, experiments were conducted to test directly whether NF-kappaB could act as a survival factor for NGF-deprived neurons. Microinjection of cells with an expression plasmid encoding NF-kappaB (c-Rel) resulted in enhanced neuronal survival after withdrawal of NGF, whereas cells that were transfected with a vector encoding a mutated derivative of c-Rel lacking the transactivation domain underwent cell death to the same extent as control cells. Together, these findings suggest that the activation of NF-kappaB/Rel transcription factors may contribute to the survival of NGF-dependent sympathetic neurons.
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PMID:Nerve growth factor-dependent activation of NF-kappaB contributes to survival of sympathetic neurons. 985 73

We characterized kinetic and biochemical changes during glucocorticoid (GC)-induced apoptosis of immature CD8+CD4+ double-positive (DP) thymocytes. A GC analog dexamethasone (Dex) induced rapid apoptotic commitment and a transient up-regulation of the NF-kappaB/RelA-p50-binding activity in DP cells. This required an early activation of proteasome, as judged by the ability of a specific proteasomal inhibitor, lactacystine, to delay apoptosis and to suppress Dex-dependent NF-kappaB activation. Dex-induced apoptotic commitment was preceded by the rapid (3 h) cleavage of both a typical caspase substrate, poly(ADP-ribose) polymerase (PARP), and of nuclear transcription factors AP-1, NF-kappaB p50-p50 and NUR-77. By contrast, phorbol myristate acetate (PMA) and/or ionomycin-induced apoptosis had much slower kinetics, were preceded by an early increase of NF-kappaB/RelA-p50, AP-1 and NUR-77 activities, and were insensitive to proteasome inhibition. Both the transgenic Bcl-2 and zVAD-fmk, an inhibitor of caspases, affected all features of Dex-induced apoptosis in a similar fashion, by inhibiting cell death and PARP cleavage, and by stabilizing AP-1, NF-kappaB p50-p50 and NUR-77 levels. Furthermore, Bcl-2 prevented Dex-induced RelA-p50 activation. However, a higher gene dosage of the transgenic Bcl-2 was required for protection against Dex, compared to the PMA and/or ionomycin-induced apoptosis. These findings highlight the unique mechanistic features of GC-induced apoptosis.
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PMID:Biochemical and kinetic characterization of the glucocorticoid-induced apoptosis of immature CD4+CD8+ thymocytes. 988 1

The transcription factor NF-kappaB is composed of homodimeric and heterodimeric complexes of Rel/NF-kappaB-family polypeptides, which include Rel-A, c-Rel, Rel-B, NF-kappaB/p50 and NF-kappaB2/p52 . The NF-kappaB1 gene encodes a larger precursor protein, p105, from which p50 is produced constitutively by proteasome-mediated removal of the p105 carboxy terminus. The p105 precursor also acts as an NFkappaB-inhibitory protein, retaining associated p50, c-Rel and Rel-A proteins in the cytoplasm through its carboxy terminus. Following cell stimulation by agonists, p105 is proteolysed more rapidly and released Rel subunits translocate into the nucleus. Here we show that TPL-2 , which is homologous to MAP-kinase-kinase kinases in its catalytic domain, forms a complex with the carboxy terminus of p105. TPL-2 was originally identified, in a carboxy-terminal-deleted form, as an oncoprotein in rats and is more than 90% identical to the human oncoprotein COT. Expression of TPL-2 results in phosphorylation and increased degradation of p105 while maintaining p50 production. This releases associated Rel subunits or p50-Rel heterodimers to generate active nuclear NF-kappaB. Furthermore, kinase-inactive TPL-2 blocks the degradation of p105 induced by tumour-necrosis factor-alpha. TPL-2 is therefore a component of a new signalling pathway that controls proteolysis of NF-kappaB1 p105.
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PMID:TPL-2 kinase regulates the proteolysis of the NF-kappaB-inhibitory protein NF-kappaB1 p105. 995 Apr 30

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor kappaB (NF-kappaB) p50 and p105 (p50/IkappaB gamma), a precursor protein of NF-kappaB p50. GFP-p50 and GFP-p105 were expressed in monkey COS-7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP-p50 (without IkappaB gamma) in the transfected cells resided in the nucleus. On the other hand, GFP-p105 (GFP-p50 with IkappaB gamma) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF-kappaB/IkappaB. In addition, the translocation of NF-kappaB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10-20 min after stimulation with hydrogen peroxide or tumor necrosis factor alpha. It was suggested that this might be due to the existence of a limiting step where NF-kappaB is released from NF-kappaB/IkappaB by the proteasome.
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PMID:Nuclear translocation of green fluorescent protein-nuclear factor kappaB with a distinct lag time in living cells. 1003 37

The basal transcription of the CXC chemokine, melanocyte growth stimulatory activity (MGSA)/growth-regulated protein (GRO)-alpha, is up-regulated in Hs294T melanoma cells compared with the normal retinal pigment epithelial (RPE) cells. Previous studies characterized a cytokine-inducible, functional nuclear factor (NF)-kappaB consensus element in the immediate 5' regulatory region of the MGSA/GRO-alpha gene at -78 bp. Although the cytokine-inducible mechanisms for transcription of this gene are fairly well delineated, the mechanisms involved in its basal up-regulation of transcription in Hs294T melanoma cells are poorly understood. Recently, we demonstrated an increased rate of IkappaB-alpha degradation in Hs294T cells, which leads to an increased nuclear localization of NF-kappaB (R. L. Shattuck-Brandt and A. Richmond. Cancer Res., 57: 3032-3039, 1997). Here we demonstrate that Hs294T melanoma cells have elevated basal IkappaB kinase (IKK) activity relative to RPE cells, causing an increased constitutive IkappaB-alpha phosphorylation and degradation. We also show here that the resultant elevated nuclear NF-kappaB (p50/p65) in these cells is responsible for the increased basal transcription of MGSA/GRO-alpha. Pretreatment of Hs294T or RPE cells with proteasome inhibitors MG115 or MG132 captures the slower migrating, constitutively phosphorylated form of IkappaB-alpha in Hs294T melanoma cells, but not in RPE cells. In addition, a phospho-specific antibody that specifically recognizes the inhibitory form of IkappaB that is phosphorylated at Ser-32 reacted with IkappaB-alpha in Hs294T cell, but not in unstimulated RPE cells. Although the basal level of protein expression of IKK-alpha or IKK-beta are the same in both Hs294T and RPE cells, immunoprecipitation with IKK-alpha antibody combined with activity assay reveal a constitutively active IKK complex in Hs294T melanoma cells. Cotransfection of a 350-bp MGSA/GRO-alpha promoter-luciferase reporter construct with either the dominant negative IKK-alpha or the repressors of NF-kappaB, the IkappaB-alpha wild type or mutants lacking the inducible phosphorylation sites, demonstrates that the increased basal MGSA/GRO-alpha transcription in the Hs294T cells is due to the enhanced nuclear activation of NF-kappaB.
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PMID:Elevated constitutive IkappaB kinase activity and IkappaB-alpha phosphorylation in Hs294T melanoma cells lead to increased basal MGSA/GRO-alpha transcription. 1009 73

The ubiquitin proteolytic system plays a major role in a variety of basic cellular processes. In the majority of these processes, the target proteins are completely degraded. In one exceptional case, generation of the p50 subunit of the transcriptional regulator NF-kappaB, the precursor protein p105 is processed in a limited manner: the N-terminal domain yields the p50 subunit, whereas the C-terminal domain is degraded. The identity of the mechanisms involved in this unique process have remained elusive. It has been shown that a Gly-rich region (GRR) at the C-terminal domain of p50 is an important processing signal. Here we show that the GRR does not interfere with conjugation of ubiquitin to p105 but probably does interfere with the processing of the ubiquitin-tagged precursor by the 26S proteasome. Structural analysis reveals that a short sequence containing a few Gly residues and a single essential Ala is sufficient to generate p50. Mechanistically, the presence of the GRR appears to stop further degradation of p50 and to stabilize the molecule. It appears that the localization of the GRR within p105 plays an important role in directing processing: transfer of the GRR within p105 or insertion of the GRR into homologous or heterologous proteins is not sufficient to promote processing in most cases, which is probably due to the requirement for an additional specific ubiquitination and/or recognition domain(s). Indeed, we have shown that amino acid residues 441 to 454 are important for processing. In particular, both Lys 441 and Lys 442 appear to serve as major ubiquitination targets, while residues 446 to 454 are independently important for processing and may serve as the ubiquitin ligase recognition motif.
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PMID:Structural motifs involved in ubiquitin-mediated processing of the NF-kappaB precursor p105: roles of the glycine-rich region and a downstream ubiquitination domain. 1020 90

HIV-1 infection of the central nervous system can cause severe neurologic disease although only microglial cells and brain macrophages are susceptible to productive viral infection. Substances secreted by infected cells are thought to cause disease indirectly. Tumor necrosis factor alpha (TNF-alpha) is one candidate neurotoxin and is upregulated during HIV-1 infection of the brain, likely via activation of the transcription factor NF-kappaB. We used the proteasome inhibitors, MG132 and ALLN (N-acetyl-Leu-Leu-Norleucinal), to inhibit NF-kappaB activation in primary human fetal microglia (PHFM) and primary monocyte derived-macrophages, and showed that they could block TNF-alpha release stimulated by lipopolysaccharide (LPS) or TNF-alpha. In addition, we performed electrophoretic mobility shift analysis and determined that in microglia, the p50/p65 heterodimer of NF-kappaB is activated by LPS stimulation, and is inhibited by MG132. Thus, blockade of NF-kappaB activation in microglia in vitro can decrease production of TNF-alpha and may prove to be a novel strategy for treating HIV-1 dementia.
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PMID:Proteasome blockers inhibit TNF-alpha release by lipopolysaccharide stimulated macrophages and microglia: implications for HIV-1 dementia. 1022 15

The NF-kappaB precursor p105 has dual functions: cytoplasmic retention of attached NF-kappaB proteins and generation of p50 by processing. It is poorly understood whether these activities of p105 are responsive to signalling processes that are known to activate NF-kappaB p50-p65. We propose a model that p105 is inducibly degraded, and that its degradation liberates sequestered NF-kappaB subunits, including its processing product p50. p50 homodimers are specifically bound by the transcription activator Bcl-3. We show that TNFalpha, IL-1beta or phorbolester (PMA) trigger rapid formation of Bcl-3-p50 complexes with the same kinetics as activation of p50-p65 complexes. TNF-alpha-induced Bcl-3-p50 formation requires proteasome activity, but is independent of p50-p65 released from IkappaBalpha, indicating a pathway that involves p105 proteolysis. The IkappaB kinases IKKalpha and IKKbeta physically interact with p105 and inducibly phosphorylate three C-terminal serines. p105 is degraded upon TNF-alpha stimulation, but only when the IKK phospho-acceptor sites are intact. Furthermore, a p105 mutant, lacking the IKK phosphorylation sites, acts as a super-repressor of IKK-induced NF-kappaB transcriptional activity. Thus, the known NF-kappaB stimuli not only cause nuclear accumulation of p50-p65 heterodimers but also of Bcl-3-p50 and perhaps further transcription activator complexes which are formed upon IKK-mediated p105 degradation.
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PMID:NF-kappaB p105 is a target of IkappaB kinases and controls signal induction of Bcl-3-p50 complexes. 1046 55


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