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)

Upon exposure to DNA-damaging agents, the p53 tumor suppressor protein is stabilized and activated, leading to cell cycle arrest, DNA repair, or apoptosis. One of the major factors that regulates the level and the transcriptional activity of p53 is the hdm2 oncoprotein. hdm2 binds to the N-terminal transactivation domain of p53 to block the transcriptional activity of p53 directly. hdm2 also functions as the E3 ligase that ubiquitinates p53 for proteasome degradation. Fluorescence anisotropy was employed to measure directly the binding of hdm2(1-126) to a p53 N-terminal peptide labeled with Oregon Green (an analogue of fluorescein). Phosphorylation of Ser15 and Ser2O did not affect the binding of the p53 peptide to hdm2. Thrl8 phosphorylation, on the other hand, reduced the binding by at least 20-fold. This suggests that phosphorylation of Thr18 could be a regulatory mechanism that disrupts the hdm2-p53 complex, thus activating p53 in response to DNA damage. The effect of p53 peptide length on binding to hdm2 was also measured quantitatively. Interestingly, p53(18-26) exhibits 10-fold higher affinity to hdm2 than do longer peptides (20- or 35-mer). This result may reflect a strong entropic barrier to binding for the longer peptides.
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PMID:Thermodynamics of p53 binding to hdm2(1-126): effects of phosphorylation and p53 peptide length. 1103 16

The wild type p53 tumor suppressor protein is rapidly degraded in normal cells by MDM2, the ubiquitin ligase that serves as the key regulator of p53 function by modulating protein stability. Cellular exposure to genotoxic stress triggers the stabilization of p53 by multiple pathways that converge upon interference with MDM2 function. In this study, we first investigated the ability of HDM2 (MDM2 human homologue) to degrade endogenous p53 in neuroblastoma (NB). Although the p53 protein in NB has been reported to be constitutively stabilized, we find that HDM2 in NB is functional and facilitates the rapid turnover of p53 in nonstressed cells via the proteasome pathway. Second, we examined the relationship between p53 and HDM2 in the adriamycin-mediated stabilization of p53 in NB. We demonstrate that while p53 stabilization depends neither upon the phosphorylation of specific N-terminal sites nor upon dissociation from HDM2, it requires inactivation of functional HDM2. In support of this notion, p53 stabilization following adriamycin resulted in an inhibition of both p53 ubiquitination and HDM2 ligase activity. Taken together, these data implicate a requirement for enzymatic inactivation of HDM2 as a novel mechanism for p53 stabilization in the DNA damage response pathway.
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PMID:Requirement for HDM2 activity in the rapid degradation of p53 in neuroblastoma. 1127 10

The glucocorticoid receptor (GR) and the tumor suppressor p53 mediate different stress responses. We have studied the mechanism of their mutual inhibition in normal endothelial cells (HUVEC) in response to hypoxia, a physiological stress, and mitomycin C, which damages DNA. Dexamethasone (Dex) stimulates the degradation of endogenous GR and p53 by the proteasome pathway in HUVEC under hypoxia and mitomycin C treatments, and also in hepatoma cells (HepG2) under normoxia. Dex inhibits the functions of p53 (apoptosis, Bax, and p21(WAF1/CIP1) expression) and GR (PEPCK and G-6-Pase expression). Endogenous p53 and GR form a ligand-dependent trimeric complex with Hdm2 in the cytoplasm. Disruption of the p53-HDM2 interaction prevents Dex-induced ubiquitylation of GR and p53. The ubiquitylation of GR requires p53, the interaction of p53 with Hdm2, and E3 ligase activity of Hdm2. These results provide a mechanistic basis for GR and p53 acting as opposing forces in the decision between cell death and survival.
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PMID:Ligand-dependent interaction of the glucocorticoid receptor with p53 enhances their degradation by Hdm2. 1156 47

The principal regulator of p53 stability is HDM2, an E3 ligase that mediates p53 degradation via the ubiquitin-26S proteasome pathway. The current model holds that p53 degradation occurs exclusively on cytoplasmic proteasomes and hence has an absolute requirement for nuclear export of p53 via the CRM-1 pathway. However, proteasomes are abundant in both cytosol and nucleus, and no studies have been done to determine under what physiological circumstances p53 degradation might occur in the nucleus. We analyzed HDM2-mediated degradation of endogenous p53 in the presence of various nuclear export inhibitors of CRM-1, including leptomycin B (LMB), a noncompetitive, specific, and fast-acting inhibitor; and HTLV1-Rex protein, a potent competitive inhibitor. We found that significant HDM2-mediated p53 degradation took place in the presence of LMB or HTLV1-Rex, indicating that endogenous p53 degradation occurs locally in the nucleus, in parallel to cytoplasmic degradation. Moreover, p53 null cells that coexpressed export-defective mutants of p53 and HDM2 retained partial competence for p53 degradation. It is important that nuclear degradation of p53 occurred during the poststress recovery phase of a p53 response, after DNA damage ceased. We propose that the capability of local p53 degradation within the nucleus provides a tighter and faster control during the down-regulatory phase, when an active p53 program needs to be turned off quickly.
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PMID:Nuclear degradation of p53 occurs during down-regulation of the p53 response after DNA damage. 1179 Jul 25

The oncoprotein hdm2 ubiquitinates p53, resulting in the rapid degradation of p53 through the ubiquitin (Ub)-proteasome pathway. Hdm2-mediated destabilization and inactivation of p53 are thought to play a critical role in a number of human cancers. We have used an in vitro enzyme assay, monitoring hdm2-catalyzed Ub transfer from preconjugated Ub-Ubc4 to p53, to identify small molecule inhibitors of this enzyme. Three chemically distinct types of inhibitors were identified this way, each with potency in the micromolar range. All three types of compounds display selective inhibition of hdm2 E3 ligase activity, with little or no effect on other Ub-using enzymes. Most strikingly, these compounds do not inhibit the autoubiquitination activity of hdm2. Steady-state analysis reveals that all three classes behave as simple reversible inhibitors of the enzyme and that they are noncompetitive with respect to both substrates, Ub-Ubc4 and p53. Studies of the effects of combinations of two inhibitory molecules on hdm2 activity indicate that the three types of compounds bind in a mutually exclusive fashion, suggesting a common binding site on hdm2 for all of these inhibitors. These compounds establish the feasibility of selectively blocking hdm2-mediated ubiquitination of p53 by small molecule inhibitors. Selective inhibitors of hdm2 E3 ligase activity could provide a novel mechanism for the development of new chemotherapeutics for the treatment of human cancers.
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PMID:Differentiation of Hdm2-mediated p53 ubiquitination and Hdm2 autoubiquitination activity by small molecular weight inhibitors. 1240 76

Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation.
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PMID:EBV-encoded EBNA-5 associates with P14ARF in extranucleolar inclusions and prolongs the survival of P14ARF-expressing cells. 1274 Sep 13

The principal regulator of p53 stability is HDM2, an E3 ligase mediating p53 degradation via the ubiquitin-26S proteasome pathway. Until recently, the accepted model held that p53 degradation occurs exclusively on cytoplasmic proteasomes, with an absolute requirement for nuclear export of p53 via the CRM1 pathway. However, 26S proteasomes are abundant in cytosol and nucleus. Using forced overexpression of HDM2 in mutant p53 tumor cells, we previously found that p53 degradation occurs in both the nucleus and the cytoplasm. p53 null cells coexpressing export-defective p53 and HDM2 retained partial competence for p53 degradation, challenging the obligatory export model. Because the ability of local nuclear destruction might add important control in switching off the p53 pathway, we now test this notion for physiological situations in untransfected cells and determine the significance of this regulation. Despite nuclear export blockade by leptomycin B and HTLV1-Rex protein, two potent CRM1 inhibitors, nuclear degradation of endogenous wild-type p53 and HDM2 occurs during down-regulation of the p53 response. This was seen in RKO and U2OS cells recovering from all major forms of DNA damage, including UV, gamma-IR, camptothecin, or cisplatinum. Moreover, significant nuclear degradation of endogenous p53 and HDM2 occurs in isolated nuclear fractions prepared from these recovering cells. Furthermore, nuclear proteasomes efficiently degrade ubiquitinated p53 in vitro. Our data indicate that in nonlethal outcomes of cellular stress, when DNA damage has been successfully repaired and the active p53 response needs to be down-regulated quickly to resume normal homeostasis, both nuclear and cytoplasmic proteasomes are recruited to efficiently degrade the elevated p53 and HDM2 protein levels. The physiological significance of local nuclear destruction lies in the fact that it adds tighter control and speed to switching the p53 pathway off.
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PMID:Nuclear and cytoplasmic degradation of endogenous p53 and HDM2 occurs during down-regulation of the p53 response after multiple types of DNA damage. 1295 68

The INK4a locus (chromosome 9p21) encodes two structurally distinct tumor-suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a major role in cell cycle control and senescence pathways. We originally identified a novel collaborator of ARF, CARF, from a two-hybrid interactive screen using p19(ARF) as bait and found that CARF interacts with ARF in the perinucleolar region and activates p53 function. In the absence of ARF, it interacts with p53 directly leading to ARF-independent enhancement of p53 function and in turn undergoes a negative feedback regulation. Very recently, we found that CARF interacts with HDM2 and undergoes degradation by an HDM2-dependent proteasome pathway. CARF may exert a vital control on p53-HDM2-p21(WAF1) pathway that is central to the cell cycle control, senescence, and DNA damage response of human cells.
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PMID:CARF binds to three members (ARF, p53, and HDM2) of the p53 tumor-suppressor pathway. 1746 Jan 93

Human cytomegalovirus (HCMV) induces serum- or density-arrested human lung (LU) cells to traverse the cell cycle, providing it with a strategy to replicate in post-mitotic cells that are its cellular substrate in vivo. HCMV infection also induces high cellular levels of p53, seemingly in contradiction to the observed cell cycle progression. This study was undertaken to examine the mechanism(s) of the increased p53 abundance. HCMV infection caused a 4-fold increase in p53 that preceded a substantial increase in p53 transcripts by more than 24 h. p53 was stabilized in HCMV-infected cells (from a half-life of less than 30 min to about 8 h) and was less sensitive to proteasome-mediated degradation. Ubiquitination of p53 in mock-infected LU cells was sensitive to inhibition by trans-4-iodo, 4'-boranyl-chalcone, consistent with HDM2-catalyzing ubiquitination of p53. In HCMV-infected cells, ubiquitination of p53 was essentially undetectable. Although HDM2 had a nuclear distribution in mock-infected LU cells, in HCMV-infected cells HDM2 was translocated to the cytoplasm beginning at 12 h and demonstrated decreased cellular abundance thereafter. HDM2 was stabilized in the HCMV-infected cells by MG132, indicating a shift from p53 to HDM2 ubiquitination. p53 demonstrated a predominantly nuclear distribution in HCMV-infected cells through 48 h, resulting in p53 and HDM2 in distinct subcellular compartments. The principal mechanism responsible for increased p53 stabilization was nuclear export and degradation of HDM2. Thus, HCMV uses a shift from p53 to HDM2 ubiquitination and destabilization to obtain protracted high levels of p53, while promoting cell cycle traverse.
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PMID:Stabilization of p53 in human cytomegalovirus-initiated cells is associated with sequestration of HDM2 and decreased p53 ubiquitination. 1769 41

We initially cloned CARF (collaborator of ARF), as a novel ARF-binding protein by a yeast interaction screen. It also interacts with p53 directly leading to ARF-independent enhancement of p53 function and in turn undergoes a negative feedback regulation. Herein we report that i) CARF interacts with HDM2 and undergoes degradation by an HDM2-dependent proteasome pathway, and ii) it acts as a transcriptional repressor of HDM2. By overexpression and silencing studies, we demonstrated that CARF exerts a vital control on the p53-HDM2-p21WAF1 pathway that is frequently altered in cancer cells.
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PMID:CARF (collaborator of ARF) interacts with HDM2: evidence for a novel regulatory feedback regulation of CARF-p53-HDM2-p21WAF1 pathway. 1829 44


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