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 proteolytic degradation of the inhibitory protein MAD3/I kappa B alpha in response to extracellular stimulation is a prerequisite step in the activation of the transcription factor NF-kappa B. Analysis of the expression of human I kappa B alpha protein in stable transfectants of mouse 70Z/3 cells shows that, as for the endogenous murine protein, exogenous I kappa B alpha is degraded in response to inducers of NF-kappa B activity, such as phorbol myristate acetate or lipopolysaccharide. In addition, pretreatment of the cells with the proteasome inhibitor N-Ac-Leu-Leu-norleucinal inhibits this ligand-induced degradation and, in agreement with previous studies, stabilizes a hyperphosphorylated form of the human I kappa B alpha protein. By expressing mutant forms of the human protein in this cell line, we have been able to delineate the sequences responsible for both the ligand-induced phosphorylation and the degradation of I kappa B alpha. Our results show that deletion of the C terminus of the I kappa B alpha molecule up to amino acid 279 abolishes constitutive but not ligand-inducible phosphorylation and inhibits ligand-inducible degradation. Further analysis reveals that the inducible phosphorylation of I kappa B alpha maps to two serines in the N terminus of the protein (residues 32 and 36) and that the mutation of either residue is sufficient to abolish ligand-induced degradation, whereas both residues must be mutated to abolish inducible phosphorylation of the protein. We propose that treatment of 70Z/3 cells with either phorbol myristate acetate or lipopolysaccharide induces a kinase activity which phosphorylates serines 32 and that these phosphorylations target the protein for rapid proteolytic degradation, possibly by the ubiquitin-26S proteasome pathway, thus allowing NF-kappa B to translocate to the nucleus and to activate gene expression.
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PMID:N- and C-terminal sequences control degradation of MAD3/I kappa B alpha in response to inducers of NF-kappa B activity. 756 83

Activation of NF-kappa B by various cellular stimuli involves the phosphorylation and subsequent degradation of its inhibitor, I kappa B alpha, although the underlying mechanism remains unclear. In the present study, the role of serine/threonine phosphatases in the regulation of I kappa B alpha phosphorylation was investigated. Our studies demonstrate that incubation of human T cells with low concentrations (approximately 1-5 nM) of calyculin A or okadaic acid, potent inhibitors of protein phosphatase type 1 (PP-1) and type 2A (PP-2A), induces the phosphorylation of I kappa B alpha even in the absence of any cellular stimulus. This action of the phosphatase inhibitors, which is associated with the activation of the RelA.p50 NF-kappa B heterodimer, is not affected by agents that block the induction of I kappa B alpha phosphorylation by tumor necrosis factor alpha (TNF-alpha). Furthermore, the phosphorylated I kappa B alpha from calyculin A-treated cells, but not that from TNF-alpha-stimulated cells, is sensitive to PP-2A in vitro, suggesting the existence of fundamental differences in the phosphorylation of I kappa B alpha induced by the two different NF-kappa B inducers. However, induction of I kappa B alpha phosphorylation by both TNF-alpha and the phosphatase inhibitors is associated with the subsequent degradation of I kappa B alpha. We further demonstrate that TNF-alpha- and calyculin A-induced I kappa B alpha degradation exhibits similar but not identical sensitivities to a proteasome inhibitor. Together, these results suggest that phosphorylation of I kappa B alpha, mediated through both the TNF-alpha-inducible and the PP-2A-opposing kinases, may serve to target I kappa B alpha for proteasome-mediated degradation.
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PMID:Activation of NF-kappa B by phosphatase inhibitors involves the phosphorylation of I kappa B alpha at phosphatase 2A-sensitive sites. 762 57

The tax gene product of the human T-cell leukemia virus type I (HTLV-I) induces the nuclear expression and biological function of the NF-kappa B/Rel family of host transcription factors although the underlying mechanism remains unclear. In the present study, we demonstrate that Tax-mediated activation of NF-kappa B/Rel can be inhibited by a proteasome inhibitor, suggesting the involvement of proteolytic reactions in this Tax-specific activation pathway. Transient transfection and reporter gene assays have revealed that Tax overrides the inhibitory function of I kappa B alpha in both F9 embryonal cells and Jurkat T cells. Moreover, Tax-mediated inactivation of I kappa B alpha requires a 16 amino acid sequence element located at the N-terminal region (amino acid 21-36) of I kappa B alpha, which is also required for tumor necrosis factor alpha-induced degradation of this inhibitory protein. We further demonstrate that the proteasome inhibitor also blocks the degradation of I kappa B alpha observed in HTLV-I-infected T cells. Interestingly, inhibition of I kappa B alpha degradation in these cells led to the accumulation of a phosphorylated form of I kappa B alpha. Together, these studies suggest that Tax activation of NF-kappa B/Rel may involve induction of phosphorylation and subsequent proteasome-mediated degradation of the inhibitor I kappa B alpha.
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PMID:Activation of NF-kappa B/Rel by Tax involves degradation of I kappa B alpha and is blocked by a proteasome inhibitor. 767 60

The 240-kDa proteasome inhibitor has been reported to be an ATP-stabilized component (CF-2) of the 26 S proteasome complex. We now report that this inhibitory factor is indistinguishable from delta-aminolevulinic acid dehydratase (ALAD), the second enzyme in the pathway of heme synthesis, based upon the following observations: 1) common sequence of the first 14 N-terminal amino acids; 2) identical migration on native and SDS-polyacrylamide gel electrophoresis; 3) identical isoelectric points of pH 7.1; 4) cross-reactivity of specific polyclonal antibodies; 5) similar dehydratase and proteasome inhibitor specific activities in both proteins; and 6) the presence of both activities in recombinant ALAD. The dual role of this protein as CF-2 in the ATP/ubiquitin-dependent pathway and in heme synthesis may be an example of "gene sharing" and explains the unexpected abundance of ALAD noted in earlier studies.
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PMID:240-kDa proteasome inhibitor (CF-2) is identical to delta-aminolevulinic acid dehydratase. 817 43

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

Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)- 1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and N-tosyl-Phe-chloromethylketone, blocked IL-1 beta induction of VCAM-1 and ICAM-1 promoter-driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-kappa B in response to IL-1 beta stimulation. In contrast, norLEU did not prevent IL-1 beta-induced nuclear translocation of NF-kappa B. The effects of norLEU were specific because it did not inhibit the IL-1 beta induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells.
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PMID:Proteasome inhibitors block VCAM-1 and ICAM-1 gene expression in endothelial cells without affecting nuclear translocation of nuclear factor-kappa B. 862 76

The levels of the tumor suppressor protein p53 are generally quite low in normal cells, due in part to its rapid turnover. Previous studies have implicated ubiquitin-dependent proteolysis in the turnover of wild-type p53 but have not established whether or not p53 is itself a substrate of the ubiquitin system. In this study, inhibitors of the 26S proteasome have been used to further explore the role of ubiquitin proteolysis in regulating p53 turnover. Increased levels of the tumor suppressor protein p53 were observed in normal cells, as well as in cells expressing the human papillomavirus 16 E6 oncoprotein, on exposure of the cells to proteasome inhibitors. Pulse-chase experiments indicated that the increased p53 levels resulted from stabilization of the protein. Furthermore, ubiquitin-p53 conjugates were detected in untreated as well as gamma-irradiated cells, indicating that ubiquitin-dependent proteolysis plays a role in the normal turnover of p53. Increased levels of the cyclin:cyclin-dependent kinase inhibitor p21, a downstream effector of p53 function, were also observed in proteasome inhibitor-treated cells, and this increase was due in part to an increase in p2l mRNA.
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PMID:In vivo ubiquitination and proteasome-mediated degradation of p53(1). 865 11

NF-kappa B is a dimeric protein that serves to initiate gene transcription in higher eukaryotic cells in response to mainly pathogenic stimuli. Its activity is controlled by a third inhibitory subunit, called I kappa B. When I kappa B is bound, NF-kappa B cannot bind to DNA or enter the nucleus but is stored in a latent cytoplasmic form. Upon stimulation of cells I kappa B is released, which allows the activation of NF-kappa B. We have analyzed the molecular mechanism underlying the removal of I kappa B-alpha. Distinct extracellular stimuli lead to a phosphorylation of I kappa B-alpha of serines 32 and 36 by a yet unidentified kinase. These modifications do not directly dissociate I kappa B from NF-kappa B but render the inhibitor highly susceptible for proteolytic degradation by, presumably, the proteasome. In this paper, we report for the first time that higher molecular mass forms of I kappa B-alpha occur under conditions that lead to a phosphorylation of I kappa B-alpha and activation of NF-kappa B. These I kappa B-alpha variants had discrete molecular masses and were most prominent in cells overexpressing I kappa B-alpha, suggesting the covalent modification of I kappa B-alpha by ubiquitin conjugation. The proteasome inhibitor Cbz-Ile-Glu(O-t-Bu)-Ala-leucinal (PSI), which stabilizes the phospho form of I kappa B-alpha, only slightly increased the amount of conjugates indicating that the conjugation of I kappa B-alpha with ubiquitin was the rate-limiting step in I kappa B-alpha degradation, and not its phosphorylation or proteolysis. Our data suggest that conjugation of I kappa B-alpha with ubiquitin is an intermediate reaction in the phosphorylation-controlled degradation of I kappa B-alpha and the subsequent activation of NF-kappa B.
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PMID:Appearance of apparently ubiquitin-conjugated I kappa B-alpha during its phosphorylation-induced degradation in intact cells. 865 51

Accumulating evidence has implicated the proteasome in the processing of protein along the major histocompatibility complex (MHC) class I presentation pathway. The availability of potent proteasome inhibitors provides an opportunity to examine the role of proteasome function in antigen presentation by MHC class I molecules to CD8+ cytotoxic T lymphocytes (CTLs). We have investigated the processing and presenting of antigenic epitopes from influenza hemagglutinin in target cells treated with the inhibitor of proteasome activity MG132. In the absence of proteasome activity, the processing and presentation of the full-length hemagglutinin was abolished, suggesting the requirement for proteasome function in the processing and presentation of the hemagglutinin glycoprotein. Epitope-containing translation products as short as 21 amino acids when expressed in target cells required proteasome activity for processing and presentation of the hemagglutin epitope to CTLs. However, when endogenous peptides of 17 amino acids or shorter were expressed in target cells, the processing and presentation of epitopes contained in these peptides were insensitive to the proteasome inhibitor. Our results support the hypothesis that proteasome activity is required for the generation of peptides presented by MHC class I molecules and that the requirement for proteasome activity is dependent on the size of the translation product expressed in the target cell. The implications of these findings are discussed.
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PMID:The requirement for proteasome activity class I major histocompatibility complex antigen presentation is dictated by the length of preprocessed antigen. 866 12

Ornithine decarboxylase (ODC) is degraded in an ATP-dependent manner in vitro by the 26 S proteasome in the presence of antizyme, an ODC destabilizing protein induced by polyamines. In the present study we examined whether the proteasome catalyses ODC degradation in living mammalian cells. Lactacystin, the most selective proteasome inhibitor, strongly inhibited the degradation of ODC that had been induced in hepatoma tissue-culture (HTC) cells by refeeding with fresh medium. Furthermore the inhibitor inhibited the rapid degradation of ODC that had been induced by hypotonic shock. Interestingly, hypertonic shock was found to increase the proportion of OD present as a complex with antizyme (the ratio of ODC-antizyme complex to total ODC). Cycloheximide, which partly inhibited rapid ODC degradation caused by hypertonic shock, also part inhibited the increase in the ratio of ODC-antizyme complex total ODC. These results suggest that a common ODC degradation pathway, namely the antizyme-dependent and 26 proteasome-catalysed ODC degradation pathway, is also operating in intact cells for osmoregulated ODC degradation.
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PMID:Proteasome pathway operates for the degradation of ornithine decarboxylase in intact cells. 869 89


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