Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.25.1 (proteasome)
 28,817 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.
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PMID:Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability. 897 27

The tumor suppressor p53 is degraded by the ubiquitin-proteasome system. p53 was polyubiquitinated in the presence of E1, UbcH5 as E2 and MDM2 oncoprotein. A ubiquitin molecule bound MDM2 through sulfhydroxy bond which is characteristic of ubiquitin ligase (E3)-ubiquitin binding. The cysteine residue in the carboxyl terminus of MDM2 was essential for the activity. These data suggest that the MDM2 protein, which is induced by p53, functions as a ubiquitin ligase, E3, in human papillomavirus-uninfected cells which do not have E6 protein.
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PMID:Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. 945 May 43

Previous studies have shown that the oncogenic HPV E6 proteins form a complex with the human homologue of the Drosophila tumour suppressor protein, discs large (Dlg). This is mediated by the carboxy terminus of the E6 proteins and involves recognition of at least one PDZ domain of Dlg. This region of E6 is not conserved amongst E6 proteins from the low risk papillomavirus types and, hence, binding of HPV E6 proteins to Dlg correlates with the oncogenic potential of these viruses. We have performed studies to investigate the consequences of the interaction between E6 and Dlg. Mutational analysis of both the HPV18 E6 and Dlg proteins has further defined the regions of E6 and Dlg necessary for complex formation. Strikingly, co-expression of wild type HPV18 E6 with Dlg in vitro or in vivo results in a dramatic decrease in the amount of Dlg protein, whereas mutants of E6 which fail to complex with Dlg have minimal effect on Dlg protein levels. The oncogenic HPV16 E6 also decreased the Dlg levels, but this was not observed with the low risk HPV11 E6 protein. Moreover, a region within the first 544 amino acids of Dlg containing the three PDZ domains confers susceptibility to E6 mediated degradation. Finally, treatment of cells with a proteasome inhibitor overrides the capacity of E6 to degrade Dlg. These results demonstrate that Dlg is targeted by high risk HPV E6 proteins for proteasome mediated degradation.
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PMID:Oncogenic human papillomavirus E6 proteins target the discs large tumour suppressor for proteasome-mediated degradation. 1052 25

Rapid degradation of wild-type p53 in the human uterine cervix is induced by the infection of high-risk human papilloma virus (HPV) types 16 and 18. HPV-E6 protein plays a critical role in the poly-ubiquitination of wild-type p53 by mediating the association of p53 with E6-associated protein (E6AP). As a result, the poly-ubiquitinated p53 is rapidly and selectively degraded by the 26S proteasome. We have established a high throughput assay system to monitor poly-ubiquitination of wild-type p53 using a new fluorescence homogeneous technology known as Homogeneous Time-Resolved Fluorescence (HTRFTM). The Europium Cryptate [Eu(K)]-labeled ubiquitins are incorporated into poly-ubiquitin chains conjugated with the biotinylated p53. In the HTRF assay, Europium cryptate-labeled ubiquitin and streptavidin-labeled allophycocyanin (XL665) are used as the fluorescence donor and acceptor, respectively. The biotinylated p53 is ubiquitinated by ubiquitination enzymes, then by the addition of streptavidin-labeled XL665, the donor and acceptor molecules are brought in close proximity, thereby generating fluorescent signals. This time-resolved fluorescence assay system shows a sufficient signal for its application in synthetic compound screening and having almost the same level of sensitivity as that monitored by the scintillation proximity assay (SPA) using 125I-labeled ubiquitin. The detection of poly-ubiquitination of wild-type p53 by using the HTRFTM or SPA systems described here is much easier and quicker than by using conventional methods. Therefore, these new systems would be appropriate for high throughput screening of compounds for the discovery of new inhibitors of poly-ubiquitination of wild-type p53.
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PMID:Application of homogeneous time-resolved fluorescence (HTRFTM) to monitor poly-ubiquitination of wild-type p53. 1053 89

Human papillomaviruses (HPVs) are associated with a number of clinical conditions, of which the most serious is cervical carcinoma. The E6 protein of the oncogenic, mucosal-specific HPV types has been shown to complex with p53 and, as a result, target it for rapid proteasome-mediated degradation. As a consequence, p53's growth-arrest and apoptosis-inducing activities are abrogated. Since p53 is frequently wild type in cervical cancers, unlike other cancers in which it is often mutated, the notion has arisen that E6's activity with respect to p53 is equivalent to an inactivating mutation of p53. In addition, several studies have shown that the pathways both upstream and downstream of p53 are intact in cervical cancers; this suggests the potential importance of the E6 - p53 interaction for therapeutic intervention. However, like all viral oncoproteins, E6 is a multifunctional protein and a plethora of other cellular targets has been identified. Indeed, E6's interactions with some of these additional targets appear to be equally important in the pathogenesis of HPV, and may also represent valid targets for therapeutic intervention.
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PMID:The role of the E6-p53 interaction in the molecular pathogenesis of HPV. 1061 9

The E6 protein of the high-risk human papillomaviruses (HPVs) and the cellular ubiquitin-protein ligase E6AP form a complex which causes the ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoproteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-ubiquitination results primarily from an intramolecular transfer of ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.
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PMID:Human papillomavirus type 16 E6 induces self-ubiquitination of the E6AP ubiquitin-protein ligase. 1086 52

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitin-activating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-to-arginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.
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PMID:Multiple C-terminal lysine residues target p53 for ubiquitin-proteasome-mediated degradation. 1104 42

In higher eukaryotic cells, the p53 protein is degraded by the ubiquitin-26S proteasome system mediated by Mdm2 or the human papilloma virus E6 protein. Here we show that COP9 signalosome (CSN)-specific phosphorylation targets human p53 to ubiquitin-26S proteasome-dependent degradation. As visualized by electron microscopy, p53 binds with high affinity to the native CSN complex. p53 interacts via its N-terminus with CSN subunit 5/Jab1 as shown by far-western and pull-down assays. The CSN-specific phosphorylation sites were mapped to the core domain of p53 including Thr155. A phosphorylated peptide, Deltap53(145-164), specifically inhibits CSN-mediated phosphorylation and p53 degradation. Curcumin, a CSN kinase inhibitor, blocks E6-dependent p53 degradation in reticulocyte lysates. Mutation of Thr155 to valine is sufficient to stabilize p53 against E6-dependent degradation in reticulocyte lysates and to reduce binding to Mdm2. The p53T155V mutant accumulates in both HeLa and HL 60 cells and exhibits a mutant (PAb 240+) conformation. It induces the cyclin-dependent inhibitor p21. In HeLa and MCF-7 cells, inhibition of CSN kinase by curcumin or Deltap53(145-164) results in accumulation of endogenous p53.
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PMID:COP9 signalosome-specific phosphorylation targets p53 to degradation by the ubiquitin system. 1128 27

It has recently been shown that the high-risk human papillomavirus (HPV) E6 proteins can target the PDZ-domain containing proteins, Dlg, MUPP-1, MAGI-1 and hScrib for proteasome-mediated degradation. However, the E6 proteins from HPV-16 and HPV-18 (the two most common high-risk virus types) differ in their ability to target these proteins in a manner that correlates with their malignant potential. To investigate the underlying mechanisms for this, we have mutated HPV-16 and HPV-18 E6s to give each protein the other's PDZ-binding motif. Analysis of these mutants shows that the greater ability of HPV-18 E6 to bind to these proteins and to target them for degradation is indeed due to a single amino acid difference. Using a number of assays, we show that the E6 proteins interact specifically with only one of the five PDZ domains of MAGI-1, and this is the first interaction described for this particular PDZ domain. We also show that the guanylate kinase homology domain and the regions of MAGI-1 downstream of amino acid 733 are not required for the degradation of MAGI-1. Finally, in a series of comparative analyses, we show that the degradation of MAGI-1 occurs through a different mechanism from that used by the E6 protein to induce the degradation of Dlg and p53.
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PMID:HPV E6 and MAGUK protein interactions: determination of the molecular basis for specific protein recognition and degradation. 1157 40

We have compared the intracellular location of the HPV E6 and E7 proteins from high- and low-risk virus types. While high-risk HPV E7 displays diffuse nuclear expression, low-risk E7 has a nuclear punctuate pattern of expression. Similarly, while high-risk E6 is expressed throughout the cell, low-risk E6 is again predominantly nuclear with a punctuate pattern of expression. Both low-risk viral oncoproteins show colocalization with PML, whereas high-risk viral proteins do not. Finally, inhibition of the proteasome pathway results in a dramatic nuclear accumulation of high-risk E6 protein. These results demonstrate fundamental differences in the localization of these viral oncoproteins within the cell and offer alternative explanations for their respective differences in pathology.
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PMID:Comparative analysis of the intracellular location of the high- and low-risk human papillomavirus oncoproteins. 1185 95


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