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 p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.
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PMID:An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo. 1082 82

The objectives of this study were (1) to assess the role of a proteasome-dependent proteolytic pathway in the pathogenesis of acute renal failure (ARF) induced by ischemia-reperfusion, and (2) to determine the involvement of this proteolytic pathway in the enhanced production of renal endothelin-1 (ET-1) in this model of ARF. ARF was induced by clamping the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. Renal function parameters such as blood urea nitrogen, plasma creatinine, creatinine clearance, urine flow, urinary osmolality and fractional excretion of sodium were measured to test the effectiveness of drugs used. Renal function in untreated ARF rats markedly decreased at 24 h after reperfusion. In addition, a marked increase in renal ET-1 content was evident in the ARF rats, compared to the sham-operated rats. Intraperitoneal injection of a proteasome inhibitor (PSI), N-benzyloxycarbonyl-Ile-Glu(O-t-Bu)-Ala-leucinal, at a dose of 1 mg/kg, 1 h before the clamping, significantly attenuated the renal function impairment in the ischemic ARF rats, and the effect was accompanied by a decrease in renal ET-1 content. On the other hand, a calpain inhibitor, calpeptin, had little effect at the same dose. These results suggest that a proteasome-dependent proteolytic pathway is involved in the enhanced production of ET-1 in the kidney and the consequent renal functional damage in ischemic ARF.
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PMID:Proteasome inhibition attenuates renal endothelin-1 production and the development of ischemic acute renal failure in rats. 1107 83

Members of the HERC (domain homologous to E6 associated protein carboxy-terminus and RCC1 domain protein) family may function both as guanine nucleotide exchange factors and E3 ubiquitin ligases. Here we identify an unstudied member, HERC3. This protein was recognized by specific antibodies in different cell types. HERC3 was located in the cytosol and in vesicular-like structures containing beta-COP, ARF and Rab5 proteins. Involvement of HERC3 in the ubiquitin system was suggested by its ability to interact with ubiquitin. The conserved cysteine in HECT proteins was not essential for this non-covalent binding. Moreover, HERC3 was a substrate of ubiquitination being degraded by the proteasome. These observations indicate a fine regulation of HERC3 and suggest a role in vesicular traffic and ubiquitin-dependent processes.
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PMID:HERC3 binding to and regulation by ubiquitin. 1116 99

Tonicity-responsive genes are regulated by the TonE enhancer element and the tonicity-responsive enhancer binding protein (TonEBP) transcription factor with which it interacts. Urea, a permeant solute coexistent with hypertonic NaCl in the mammalian renal medulla, activates a characteristic set of signaling events that may serve to counteract the effects of NaCl in some contexts. Urea inhibited the ability of hypertonic stressors to increase expression of TonEBP mRNA and also inhibited tonicity-inducible TonE-dependent reporter gene activity. The permeant solute glycerol failed to reproduce these effects, as did cell activators including peptide mitogens and phorbol ester. The inhibitory effect of urea was evident as late as 2 h after the application of hypertonicity. Pharmacological inhibitors of known urea-inducible signaling pathways failed to abolish the inhibitory effect of urea. TonEBP action is incompletely understood, but evidence supports a role for proteasome function and p38 action in regulation; urea failed to inhibit proteasome function or p38 signaling in response to hypertonicity. Consistent with its effect on TonEBP expression and action, urea pretreatment inhibited the effect of hypertonicity on expression of the physiological effector gene, aldose reductase. Taken together, these data 1) define a molecular mechanism of urea-mediated inhibition of tonicity-dependent signaling, and 2) underscore a role for TonEBP abundance in regulating TonE-mediated gene transcription.
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PMID:Urea inhibits hypertonicity-inducible TonEBP expression and action. 1129 34

The structure--function relationships occurring on the bovine thymus 20S proteasome, which exhibits the features of an immunoproteasome, have been studied. The investigation has been performed, essentially, using a fluorimetric approach, taking advantage either of the sensitivity of the complex to sodium dodecil sulfate and chaotropic agents (urea and guanidine hydrochloride) or of the presence, on the molecule, of a high number of tryptophan residues. The results obtained indicate that the perturbation or the oxidation of these residues affect the catalytic events taking place on the thymus proteasome and that the functional effects determined by SDS and chaotropic agents most likely occur through a series of progressive structural modifications leading to an inactive molecule. The presence of structural intermediates in the proteasome inactivation process suggests that thymus proteasome is a molecule characterized, at the same time, by structural flexibility (modulation of active sites) and structural stability (maintaining of the quaternary structure) in agreement with its crucial role in the cell life cycle.
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PMID:Structure--function relationships in bovine thymus 20S proteasome: a fluorimetric study. 1131 22

While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14(ARF), Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.
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PMID:The corepressor mSin3a interacts with the proline-rich domain of p53 and protects p53 from proteasome-mediated degradation. 1135 5

The 20S proteasome from the extreme thermophile Methanococcus jannaschii (Mj) was purified and sequenced to facilitate production of the recombinant proteasome in E. coli. The recombinant proteasome remained in solution at a purity level of 80-85% (according to SDS PAGE) following incubation of cell lysates at 70 degrees C. Temperature-activity profiles indicated that the temperature optima of the wild-type and recombinant enzymes differed substantially, with optimal activities occurring at 119 degrees C and 95 degrees C, respectively. To ameliorate this discrepancy, two recombinant enzyme preparations were produced, each of which included denaturation of the proteasome by 4 M urea followed by high-temperature (85 degrees C) dialysis. The wild-type temperature optimum was restored, but only if proteasome subunits were denatured and refolded prior to assembly (a preparation designated as alpha & beta). In contrast, when proteasome assembly preceded denaturation (designated alpha + beta) the optimum temperature was raised to a lesser degree. Moreover, the alpha & beta and alpha + beta preparations had apparent thermal half-lives at 114 degrees C of 54.2 and 26.2 min, respectively, and the thermostability of the less stable enzyme was more sensitive to a reduction in pH. Attainment of wild-type activity and stability thus required the proper folding of both the alpha- and beta-subunits prior to proteasome assembly. Consistent with this behavior, dual-scanning calorimetry (DSC) measurements revealed differences in the reassembly efficiency of the two proteasome preparations. The ability to produce structural conformers with dramatically different thermal optima and thermostabilities may facilitate the determination of molecular forces and structural motifs responsible for enzyme thermostablity and high-temperature activity.
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PMID:Chemical denaturation and elevated folding temperatures are required for wild-type activity and stability of recombinant Methanococcus jannaschii 20S proteasome. 1151 79

The tumor suppressor p53 is a labile protein whose level is known to be regulated by the Mdm-2-ubiquitin-proteasome degradation pathway. We have found another pathway for p53 proteasomal degradation regulated by NAD(P)H quinone oxidoreductase 1 (NQO1). Inhibition of NQO1 activity by dicoumarol induces p53 and p73 proteasomal degradation. A mutant p53 (p53([22,23])), which is resistant to Mdm-2-mediated degradation, was susceptible to dicoumarol-induced degradation. This finding indicates that the NQO1-regulated proteasomal p53 degradation is Mdm-2-independent. The tumor suppressor p14(ARF) and the viral oncogenes SV40 LT and adenovirus E1A that are known to stabilize p53 inhibited dicoumarol-induced p53 degradation. Unlike Mdm-2-mediated degradation, the NQO1-regulated p53 degradation pathway was not associated with accumulation of ubiquitin-conjugated p53. In vitro studies indicate that dicoumarol-induced p53 degradation was ubiquitin-independent and ATP-dependent. Inhibition of NQO1 activity in cells with a temperature-sensitive E1 ubiquitin-activating enzyme induced p53 degradation and inhibited apoptosis at the restrictive temperature without ubiquitination. Mdm-2 failed to induce p53 degradation under these conditions. Our results establish a Mdm-2- and ubiquitin-independent mechanism for proteasomal degradation of p53 that is regulated by NQO1. The lack of NQO1 activity that stabilizes a tumor suppressor such as p53 can explain why humans carrying a polymorphic inactive NQO1 are more susceptible to tumor development.
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PMID:Mdm-2 and ubiquitin-independent p53 proteasomal degradation regulated by NQO1. 1223 53

The 97-kDa valosin-containing protein (p97-VCP or VCP), a hexameric AAA ATPase, plays an important role in diverse cell activities, including ubiquitin-proteasome mediated protein degradation. In this report, we studied dissociation-reassembly kinetics to analyze the structure-function relationship in VCP. Urea-dissociated VCP can reassemble by itself, but addition of ATP, ADP, or ATP-gamma S accelerates the reassembly. Mutation in the ATP-binding site of D1, but not D2, domain abolishes the ATP acceleration effect and further delays the reassembly. Using hybrid hexamers of the wild type and ATP-binding site mutant, we show that hexameric structure and proper communication among the subunits are required for the ATPase activity and ubiquitin-proteasome mediated degradation. Thus, ATP-binding site in D1 plays a major role in VCP hexamerization, of which proper inter-subunit interaction is essential for the activities.
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PMID:Hexamerization of p97-VCP is promoted by ATP binding to the D1 domain and required for ATPase and biological activities. 1250 76

In a 5L fermentor the production conditions of alkaline protease gene engineering strain BA071 were investigated. The maximum activity of alkaline protease reached 24,480 u/mL in 40 hours of fermentation by combination of enhancing aeration and changing the agitation rate. The fast purification method of recombinant protease was conducted with FPLC (Fast Protein Liquid Chromatography). The crude enzyme, treated with ammonium sulfate fractionation and decolored with DEAE-A-50 and polyethylene glycol concentration, was purified with CM-Sephadex-C-50 and Sephadex-G-75. The purified enzyme appears homologous on SDS-PAGE. The purity of enzyme was increased 76.2 times. SDS-PAGE analysis showed that the molecular weights of expressed recombinant products were about 28 kD. The optimal reaction pH and temperature of recombinant enzyme were at pH11 and 60 degrees C, respectively. The recombinant enzyme exhibited high temperature tolerance and was stable at a wide range of pH. Ca2+, MG2+ can enhance the stability of the recombinant enzyme. While the protease activity of the enzyme was strongly inhibited by Hg2+, Ag+, PMFS [symbol: see text] DFP, and was not affected by SDS and Urea.
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PMID:[Study on fermentation condition of alkaline protease gene engineering strain and the purification and characterization of recombinant enzyme]. 1267 45


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