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 tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.
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PMID:Regulation of p53 stability by Mdm2. 915 96

Upon activation in response to cellular stress or DNA damage, the p53 tumor suppressor induces the expression of gene products involved in cell cycle arrest and apoptosis. Using the proteasome-specific inhibitors, MG132 (N-acetyl-L-leucinyl-L-leucinal-L-leucinal) and lactacystin, here we show that the p53-response proteins, bax and mdm2 as well as p21, are degraded by the ubiquitin-proteasome pathway in HeLa cells. MG132 also increased expression of the three proteins in cells that lack p53, showing that stabilization of the p53 response proteins is not due to increased levels of p53 itself. Increases in mdm2 protein levels by MG132 was accompanied by increases in polyubiquitinated forms of the proteins. Our results indicate that ubiquitin-dependent protein degradation influences the turnover of downstream targets of p53, therefore suggesting that the proteasome plays a role in regulating apoptosis and cell cycle arrest in response to p53.
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PMID:mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway. 943 91

The Hdm2 oncoprotein inhibits p53 functions by two means: (i) it blocks p53's transactivation activity and (ii) it targets p53 for degradation in a proteasome-dependent manner. Recent data indicate that Hdm2 shuttles between the nucleus and the cytoplasm and that the regulation of p53 levels by Hdm2 requires its nuclear export activity. Two different models are consistent with these observations. In the first, Hdm2 binds to p53 in the nucleus and shuttles p53 from the nucleus to the cytoplasm, and then it targets p53 to the cytoplasmic proteasome. Alternatively, Hdm2 and p53 could be exported separately from the nucleus and then associate in the cytoplasm, where Hdm2 promotes the degradation of p53. To distinguish between these two models, several Hdm2 mutants were employed. Hdm2NLS lacks the ability to enter the nucleus, whereas Hdm2NES is deficient in nuclear export. Hdm2NLS, Hdm2NES, or the combination of both mutants were unable to promote p53 degradation in the cotransfected 2KO cells (which were null for both the p53 and mdm2 genes), although wild-type Hdm2 efficiently reduced p53 levels under the same conditions. This observation is not a result of the differences in expression levels or stability between Hdm2 and these mutants. Moreover, coexpression of these mutants had no effect on wild-type Hdm-2-induced p53 destabilization. Thus, Hdm2 must shuttle p53 from the nucleus to the cytoplasm to target it for degradation in the cytoplasm.
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PMID:Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. 1007 39

Regulation of the stability of p53 is key to its tumor-suppressing activities. mdm2 directly binds to the amino-terminal region of p53 and targets it for degradation through the ubiquitin-proteasome pathway. The coactivator protein TAF(II)31 binds to p53 at the amino-terminal region that is also required for interaction with mdm2. In this report, we demonstrate that expression of TAF(II)31 inhibits mdm2-mediated ubiquitination of p53 and increases p53 levels. TAF(II)31-mediated p53 stabilization results in activation of p53-mediated transcriptional activity and leads to p53-dependent growth arrest in fibroblasts. UV-induced stabilization of p53 coincides with an increase in p53-associated TAF(II)31 and a corresponding decrease in mdm2-p53 interaction. Non-p53 binding mutant of TAF(II)31 fails to stabilize p53. Our results suggest that direct interaction of TAF(II)31 and p53 not only mediates p53 transcriptional activation but also protects p53 from mdm2-mediated degradation, thereby resulting in activation of p53 functions.
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PMID:Stabilization and activation of p53 by the coactivator protein TAFII31. 1127 72

The mdm2 gene product is an important regulator of p53 function and stability. mdm2 is an E3 ubiquitin ligase for p53 and the RING finger domain of mdm2 is critical for ligase activity. Ubiquitin (Ub) conjugation is a general targeting modification and poly-ubiquitin chains specifically target proteins to the proteasome for degradation. In this report, we show that the multistep cascade of mdm2-mediated p53 ubiquitination can be reduced to three purified recombinant proteins: ubiquitin-conjugated E2, mdm2, and p53. This simplification allows enzymatic analysis of the isolated ligase reaction. The simplified reaction recapitulates the ubiquitination of p53 observed with individual components and the p53-Ub((n)) is qualitatively similar to p53-Ub((n)) detected in lactacystin-treated cells. Surprisingly, we find that p53 is modified with multiple mono-ubiquitin moieties as opposed to a poly-ubiquitin chain. Finally, kinetic analysis indicates the transfer reaction proceeds either through a modified Ping Pong mechanism involving requisite enzyme isomerization steps, or through a Rapid Equilibrium Random Bi Bi mechanism involving very large anti-cooperative interactions between the two substrate binding pockets on the enzyme, mediated through allosteric changes in enzyme structure.
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PMID:Human mdm2 mediates multiple mono-ubiquitination of p53 by a mechanism requiring enzyme isomerization. 1139 92

The p53 tumor suppressor is stabilized in cells expressing the human papillomavirus type 16 (HPV-16) E7 oncoprotein. In contrast, expression of the HPV-16 E6 protein inactivates p53 by targeting it for proteasomal degradation. Since p53 activation is associated with protein accumulation we investigated the biochemical mechanisms and biological consequences of p53 stabilization in HPV-16 E7-expressing cells. Transcriptional reporter assays, expression profiling studies using cDNA arrays, and immunoblot analyses of known p53 target genes suggest that p53 remains transcriptionally inert in E7-expressing cells. The stabilized p53 in E7-expressing cells is in a wild-type conformation and the same number of phospho-forms is present. Furthermore, E7 expression does not alter p53 localization or generally block nuclear export or proteasomal degradation of p53. Moreover, the stabilized p53 remains susceptible to mdm2-induced proteasome-mediated degradation, and exogenous transfected p53 is transcriptionally active in E7-expressing cells. Taken together, these results suggest that E7 can interfere with the normal turnover of p53 but that the resulting increase of p53 has no detectable transcriptional consequences on the p53 targets that we investigated.
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PMID:Stabilization and functional impairment of the tumor suppressor p53 by the human papillomavirus type 16 E7 oncoprotein. 1203 67

Advanced prostate cancer is resistant to current therapeutic strategies. Bortezomib (Velcade; previously called PS-341) is a potent and specific inhibitor of the 26S proteasome that is currently in clinical trials for treatment of various malignancies, including prostate cancer. We investigated the effects of bortezomib on p53 in the LNCaP-Pro5 prostate cancer cell line. Bortezomib induced strong stabilization of p53, but it did not promote phosphorylation on serines 15 and 20, and p53 remained bound to its inhibitor, mdm2. Nonetheless, bortezomib stimulated p53 translocation to the nucleus (not mitochondria) and enhanced p53 DNA binding, accumulation of p53-dependent transcripts, and activation of a p53-responsive reporter gene. Furthermore, stable LNCaP-Pro5 transfectants of LNCaP-Pro5 expressing the p53 inhibitor human papillomavirus-E6 displayed reduced bortezomib-induced p53 activation and cell death. Together, our data demonstrate that bortezomib stimulates p53 activation via a novel mechanism.
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PMID:The proteasome inhibitor bortezomib stabilizes a novel active form of p53 in human LNCaP-Pro5 prostate cancer cells. 1461 32

Mdm2 is a negative regulator of p53 activity and functions as an E3 ubiquitin ligase of p53. Inhibition of mdm2 E3 ligase activity will block ubiquitination and subsequent proteasome-mediated degradation of p53, resulting in the stabilization of p53 protein that could lead to the restoration of its tumor-suppressor activity. This chapter describes quantitative biochemical assays for mdm2 E3 activity that can be applied to other ubiquitin-utilizing enzyme systems. Our unique assay format relies on the generation of labeled Ub-E2 conjugate that functions as a substrate for the E3 ligase enzyme. Reducing the E1-E2-E3 ubiquitin cascade to a single enzyme (E3) and bisubstrate (Ub-E2 and target protein) reaction makes it possible to carry out detailed biochemical characterization of the reaction mechanism, high-throughput screening to identify inhibitors of specific E3 ligases, and detailed characterization of the mode of inhibitor interactions with the target enzyme. In addition, preforming the Ub-E2 conjugate as an enzyme substrate for inhibitor screening minimizes interference from thiol-modifying compounds and from nucleotide analogs and other ATP-interfering compounds that might affect the E1 reaction. Using this type of format, we were able to identify small molecule inhibitors of mdm2 E3 ligase activity that are selective against E1 and other E3 ligases, including mdm2's own autoubiquitination activity. Detailed protocols on the labeling of Ub, the generation of Ub-E2, and the use of Ub-E2 in the E3 ligase reaction for inhibitor discovery and characterization are provided.
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PMID:Quantitative assays of Mdm2 ubiquitin ligase activity and other ubiquitin-utilizing enzymes for inhibitor discovery. 1633 90

Levels of the p53 tumor suppressor protein are increased in human cytomegalovirus (HCMV)-infected cells and may be important for HCMV pathogenesis. In normal cells p53 levels are kept low due to an autoregulatory feedback loop where p53 activates the transcription of mdm2 and mdm2 binds and ubiquitinates p53, targeting p53 for proteasomal degradation. Here we report that, in contrast to uninfected cells, mdm2 was undetectable upon treatment of infected fibroblasts with the proteasome inhibitor MG132. Cellular depletion of mdm2 was reproducible in p53-null cells transfected with the HCMV IE2-86 protein, but not with IE172, independently of the endogenous mdm2 promoter. IE2-86 also prevented the emergence of presumably ubiquitinated species of p53. The regions of IE2-86 important for mdm2 depletion were those containing the sequences corresponding to the putative zinc finger and C-terminal acidic motifs. mdm2 and IE2-86 coimmunoprecipitated in transfected and infected cell lysates and in a cell-free system. IE2-86 blocked mdm2's p53-independent transactivation of the cyclin A promoter in transient-transfection experiments. Pulse-chase experiments revealed that IE2-86 but not IE1-72 or several loss-of-function IE2-86 mutants increased the half-life of p53 and reduced the half-life of mdm2. Short interfering RNA-mediated depletion of IE2-86 restored the ability of HCMV-infected cells to accumulate mdm2 in response to proteasome inhibition. Taken together, the data suggest that specific interactions between IE2-86 and mdm2 cause proteasome-independent degradation of mdm2 and that this may be important for the accumulation of p53 in HCMV-infected cells.
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PMID:Evidence that the human cytomegalovirus IE2-86 protein binds mdm2 and facilitates mdm2 degradation. 1657

The stable accumulation of p53 is detrimental to the cell because it blocks cell growth and division. Therefore, increases in p53 levels are tightly regulated, mainly by its transcriptional target, mdm2, that downregulates p53. Elucidation of new signaling pathways requires the characterization of the members and the nature of their connection. Vaccinia-related kinase 1 (VRK1) contributes to p53 stabilization by partly interfering with its mdm2-mediated degradation, among other mechanisms; therefore, it is likely that some form of autoregulation between VRK1 and p53 must occur. We report here the identification of an autoregulatory loop between p53 and its stabilizing VRK1. There is an inverse correlation between VRK1 and p53 levels in cell lines, and induction of p53 by UV light downregulates VRK1 in fibroblasts. As the amount of p53 protein increases, there is a downregulation of the VRK1 protein level independent of its promoter. This effect is indirect but requires a transcriptionally active p53. The three most common transcriptionally inactive mutations detected in hereditary (Li-Fraumeni syndrome) and sporadic human cancer, p53(R175H), p53(R248W), and p53(R273H), as well as p53(R280K), are unable to induce downregulation of VRK1 protein. The p53 isoforms Delta40p53 and p53beta, lacking the transactivation and oligomerization domains, respectively, do not downregulate VRK1. VRK1 downregulation induced by p53 is independent of mdm2 activity and proteasome-mediated degradation since it occurs in the presence of proteasome inhibitors and in mdm2-deficient cells. The degradation of VRK1 is sensitive to chloroquine, an inhibitor of the late endosome-lysosome transport, and to serine protease inhibitors of the lysosomal pathway.
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PMID:p53 downregulates its activating vaccinia-related kinase 1, forming a new autoregulatory loop. 1678 68


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