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)

In MCF-7 (estrogen receptor (ER)+) and in MDA-MB-231 (ER-) cells stably transfected with either estrogen receptor alpha (ERalpha) or beta (ERbeta) subtype (MDA-MB-231 stably transfected with the mouse ERalpha cDNA (MERA) and MDA-MB-231 stably transfected with the human ERbeta cDNA (HERB), respectively) N-term heat shock protein of 90kDa (hsp90) ligands (geldanamycin and radicicol) and C-term hsp90 ligands (novobiocin) decrease the basal and estradiol (E(2))-induced transcription activity of ER on an estrogen responsive element (ERE)-LUC reporter construct concomitantly with or 1h after E(2) treatment. All hsp90 ligands induced an E(2)- and MG132-inhibited decrease of both ER cell content. However, the kinetics of these degradations are slower than those induced by the selective estrogen receptor down-regulator RU 58668 (RU). This suggests that inhibition of the hsp90 ATPase activity targets both ERs to the 26S proteasome and that hsp90 interacts with both ER subtypes. Rapamycin (Rapa) and cyclosporin A (CsA), ligands of immunophilins FK506 binding protein (FKBP52) and cyclophilin of 40kDa (CYP40) interacting in separate ER-hsp90 complexes, both induced a proteasomal-mediated degradation of ERs but not of their cognate immunophilin. Moreover, they also decrease the E(2)-induced luciferase transcription but weaker than RU and hsp90 ligands. Fluorescence activated cell sorter (FACS) analysis revealed a blockade of cell progression by RU and 4-hydroxy-tamoxifen at the G(1) phase of the cell cycle and an induction of apoptosis in MCF-7 cells. Rapa and mainly CsA (but not FK506) and hsp90 ligands promote by their own apoptosis in MCF-7, in MERA, and in HERB cells and in MDA-MB-231 ER-null cells. These data suggest that (1) hsp90, as for all steroid receptors, acts as a molecular chaperone for ERbeta; (2) ER-ligands (except tamoxifen), hsp90- and immunophilin-ligands (except FK506) target the two ER subtypes to a proteasome-mediated proteolysis via different signalling pathways; (3) hsp90- and immunophilin-ligands Rapa and CsA, alone or in association with anti-estrogens such as RU, may constitute a potential therapeutic strategy for breast cancer treatment.
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PMID:Estrogen receptor alpha and beta subtype expression and transactivation capacity are differentially affected by receptor-, hsp90- and immunophilin-ligands in human breast cancer cells. 1586 52

Small molecules designed to specifically activate or inactivate protein functions have been useful to study biological processes. PROTACS are small molecule chimera which comprise a ligand and a peptide recognition motif for an E3 ligase. These novel reagents exploit the ubiquitin-mediated proteasome degradation pathway to target the ligand-bound protein for intracellular degradation. Here, we report that an estrogen receptor (ER)-targeting PROTACS that causes degradation of ER is able to potently inhibit endothelial cell differentiation in a three-dimensional angiogenic sprouting assay. These findings support the use of ER-targeting PROTACS as probes of angiogenesis.
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PMID:Use of PROTACS as molecular probes of angiogenesis. 1587 33

In estrogen target cells, estrogen receptor-alpha (ERalpha) protein levels are strictly regulated. Although receptor turnover is a continuous process, dynamic fluctuations in receptor levels, mediated primarily by the ubiquitin-proteasome pathway, occur in response to changing cellular conditions. In the absence of ligand, ERalpha is sequestered within a stable chaperone protein complex consisting of heat shock protein 90 (Hsp90) and cochaperones. However, the molecular mechanism(s) regulating ERalpha stability and turnover remain undefined. One potential mechanism involves CHIP, the carboxyl terminus of Hsc70-interacting protein, previously shown to target Hsp90-interacting proteins for ubiquitination and proteasomal degradation. In the present study, a role for CHIP in ERalpha protein degradation was investigated. In ER-negative HeLa cells transfected with ERalpha and CHIP, ERalpha proteasomal degradation increased, whereas ERalpha-mediated gene transcription decreased. In contrast, CHIP depletion by small interference RNA resulted in increased ERalpha accumulation and reporter gene transactivation. Transfection of mutant CHIP constructs demonstrated that both the U-box (containing ubiquitin ligase activity) and the tetratricopeptide repeat (TPR, essential for chaperone binding) domains within CHIP are required for CHIP-mediated ERalpha down-regulation. In addition, coimmunoprecipitation assays demonstrated that ERalpha and CHIP associate through the CHIP TPR domain. In ERalpha-positive breast cancer MCF7 cells, CHIP overexpression resulted in decreased levels of endogenous ERalpha protein and attenuation of ERalpha-mediated gene expression. Furthermore, the ERalpha-CHIP interaction was stimulated by the Hsp90 inhibitor geldanamycin (GA), resulting in enhanced ERalpha degradation; this GA effect was further augmented by CHIP overexpression but was abolished by CHIP depletion. Finally, ERalpha dissociation from CHIP by various ERalpha ligands, including 17beta-estradiol, 4-hydroxytamoxifen, and ICI 182,780, interrupted CHIP-mediated ERalpha degradation. These results demonstrate a role for CHIP in both basal and GA-induced ERalpha degradation. Furthermore, based on our observations that CHIP promotes ERalpha degradation and attenuates receptor-mediated gene transcription, we suggest that CHIP, by modulating ERalpha stability, contributes to the regulation of functional receptor levels, and thus hormone responsiveness, in estrogen target cells.
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PMID:CHIP (carboxyl terminus of Hsc70-interacting protein) promotes basal and geldanamycin-induced degradation of estrogen receptor-alpha. 1603 32

Estrogen promotes the proliferation of human breast epithelial cells by interacting with the estrogen receptor (ER). Physiological responses of cells to estrogen are regulated in part by degradation of the ER. Previous studies revealed that calmodulin binds directly to the ER, thereby enhancing its stability. Consistent with these findings, cell-permeable calmodulin antagonists dramatically reduced the number of ER in MCF-7 human breast epithelial cells. Here we investigated the molecular mechanism by which calmodulin attenuates ER degradation. MG132 and lactacystin, inhibitors of the ubiquitin-proteasome pathway, prevented the calmodulin antagonist CGS9343B from reducing the amount of ER in MCF-7 cells. In contrast, protease inhibitors afforded no protection. Moreover, CGS9343B enhanced ER ubiquitination. A point mutant ER construct that is unable to bind calmodulin, termed ERDeltaCaM, is ubiquitinated to a greater extent than wild type ER. The ubiquitin-protein isopeptide ligase E6-associated protein (E6AP) associated with and promoted the degradation of ER. The possible convergence of calmodulin and E6AP on ER degradation was examined. ERDeltaCaM bound E6AP with higher affinity than that of wild type ER. Moreover, calmodulin attenuated the in vitro interaction between ER and E6AP in a Ca(2+)-dependent manner. Collectively, our data reveal that E6AP is a component of ER degradation via the ubiquitin-proteasome pathway and that Ca(2+)/calmodulin modulates this degradation mechanism. These results have potential implications for the development of selectively targeted therapeutic agents for breast cancer.
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PMID:E6AP and calmodulin reciprocally regulate estrogen receptor stability. 1631 11

The antiestrogen fulvestrant (ICI 182,780) causes immobilization of estrogen receptor-alpha (ERalpha) in the nuclear matrix accompanied by rapid degradation by the ubiquitin-proteasome pathway. In this study we tested the hypothesis that fulvestrant induces specific nuclear matrix protein-ERalpha interactions that mediate receptor immobilization and turnover. A glutathione S-transferase (GST)-ERalpha-activating function-2 (AF2) fusion protein was used to isolate and purify receptor-interacting proteins in cell lysates prepared from human MCF-7 breast cancer cells. After SDS-PAGE and gel excision, mass spectrometry was used to identify two major ERalpha-interacting proteins, cytokeratins 8 and 18 (CK8.CK18). We determined, using ERalpha-activating function-2 mutants, that helix 12 (H12) of ERalpha, but not its F domain, is essential for fulvestrant-induced ERalpha-CK8 and CK18 interactions. To investigate the in vivo role of H12 in fulvestrant-induced ERalpha immobilization/degradation, transient transfection assays were performed using wild type ERalpha,ERalpha with a mutated H12, and ERalpha with a deleted F domain. Of those, only the ERalpha H12 mutant was resistant to fulvestrant-induced immobilization to the nuclear matrix and protein degradation. Fulvestrant treatment caused ERalpha degradation in CK8.CK18-positive human breast cancer cells, and CK8 and CK18 depletion by small interference RNAs partially blocked fulvestrant-induced receptor degradation. Furthermore, fulvestrant-induced ERalpha degradation was not observed in CK8 or CK18-negative cancer cells, suggesting that these two intermediate filament proteins are necessary for fulvestrant-induced receptor turnover. Using an ERalpha-green fluorescent protein construct in fluorescence microscopy revealed that fulvestrant-induced cytoplasmic localization of newly synthesized receptor is mediated by its interaction with CK8 and CK18. In summary, this study provides the first direct evidence linking ERalpha immobilization and degradation to the nuclear matrix. We suggest that fulvestrant induces ERalpha to interact with CK8 and CK18, drawing the receptor into close proximity to nuclear matrix-associated proteasomes that facilitate ERalpha turnover.
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PMID:Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha. 1645 37

The eukaryotic proteasome is a 26S ATP-dependent proteolytic complex, which possesses chymotrypsin-like, trypsin-like and peptidyl glutamyl peptide hydrolase (PGPH) activities, which enable the proteasome to degrade all short-lived and many long-lived proteins, and consequently regulate a myriad of activities in cells. In this study, we observed that inhibition of the proteasome, and more specifically, inhibition of the chymotrypsin-like activity of the proteasome, in MCF-7 human breast cancer cells resulted in selective down-regulation of the nuclear estrogen receptor-alpha (ERalpha). Our data indicated that estrogen had no effect, whereas the ERalpha antagonist, tamoxifen, reduced the amount of ERalpha that could be subjected to down-regulation after proteasome inhibition. Furthermore, our data demonstrated that protein synthesis was required for the down-regulation of ERalpha to occur. Collectively, these data indicate the existence of a proteasome-dependent mechanism that is utilized by MCF-7 cells to maintain a steady-state level of ERalpha.
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PMID:Down-regulation of estrogen receptor-alpha in MCF-7 human breast cancer cells after proteasome inhibition. 1680 88

Treatment of cells with estrogens and several pure ERalpha antagonists rapidly induces down-regulation of the alpha-type estrogen receptor (ERalpha) in the nucleus by mechanisms that are sensitive to the proteasome inhibitors, MG132 and clasto-lactacystin-beta-lactone. Hence, it is believed that these ER ligands induce down-regulation of ERalpha by proteasome-dependent mechanisms, which serve to control both the amount of transcriptional activity and the level of ligand-bound ERalpha in cells. In this study, we observed that treatment of cultured MCF-7 and T47D human breast cancer cells with the low affinity ER ligand, 4,4'-dihydroxy-trans-stilbene (4,4'-DHS), inhibited the transcriptional activity of ERalpha and induced slow and gradual decrease in the amount of ERalpha protein (henceforth referred to as down-regulation of ERalpha). The 4,4'-DHS-induced down-regulation of ERalpha in MCF-7 cells involved a mechanism that was insensitive to the two most specific proteasome inhibitors, clasto-lactacystin-beta-lactone and epoxomycin, but sensitive to MG132 at concentrations exceeding that required for maximal inhibition of the proteasome in MCF-7 cells. Therefore, 4,4'-DHS appears to induce down-regulation of ERalpha by a proteasome-independent mechanism. Here, we present data to show that both 4-OH and 4'-OH are critical for the ability of 4,4'-DHS to induce down-regulation of ERalpha and suggest that 4,4'-DHS provides a useful scaffold for development of novel ERalpha antagonists.
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PMID:Proteasome-independent down-regulation of estrogen receptor-alpha (ERalpha) in breast cancer cells treated with 4,4'-dihydroxy-trans-stilbene. 1682 79

Estrogens are essential for human health. Their physiological effects are primarily mediated by two types of intracellular estrogen receptors (ER alpha and ER beta) that function as DNA-binding transcription factors. However, estrogens are also involved in the development and progression of breast cancers. Endocrine therapy aims to reduce the availability of the hormone or to counteract its action. This can be achieved by preventing estrogen production or administrating antiestrogens (AEs), synthetic drugs belonging to several distinct structural categories. Selective estrogen receptor modulators (or SERMs) bind ERs but have a mixed agonist/antagonist profile. Selective estrogen receptor downregulators (or SERDs) are pure antiestrogens, acting by decreasing the level of ERs through their ubiquitinylation and subsequent targeting to the proteasome. We review most of the usual antiestrogenic therapies for estrogen-dependent and estrogen-independent solid cancers and present our recent results on the use of AEs against multiple myeloma. In breast cancer treatments, the most commonly used antiestrogen, tamoxifen, has major limitations: side effects due to its partial agonist activity and constitutive or acquired resistance. We propose that novel AE drug delivery systems may enhance the overall beneficial effects by targeting tumoral cells.
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PMID:Antiestrogenic therapies in solid cancers and multiple myeloma. 1690 Jun 59

The ubiquitin-proteasome system plays an important role in a variety of cellular functions by means of its proteolytic activity. Interestingly, recent studies have indicated that the proteasome components are also integral parts of transcription complexes. In genome-wide screening for steroid receptor coactivator (SRC)-interacting proteins using yeast two-hybrid system, we found that the 20S proteasome beta subunit LMP2 (Low Molecular mass Polypeptide 2) interacts directly with the SRC coactivators. We showed that LMP2 is required for estrogen receptor (ER)-mediated gene transcription and for estrogen-stimulated cell cycle progression. We found that LMP2-associated proteasome is recruited to the entire sequence of ER target genes, implicating a role for the proteasome in both transcription initiation and elongation. We demonstrated that the recruitment of LMP2 by SRC coactivators is necessary for cyclic association of ER-regulated transcription complexes on ER targets. These results revealed a mechanism by which the proteasome machinery is recruited in ER-mediated gene transcription. Our experiments also provided evidence implicating SRC coactivators in gene transcription elongation.
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PMID:The catalytic subunit of the proteasome is engaged in the entire process of estrogen receptor-regulated transcription. 1695 78

Mechanisms controlling nuclear hormone receptors are a central question to mammalian developmental and disease processes. Herein, we show that a subtle increase in O-GlcNAc levels inhibits activation of nuclear hormone receptors. In vivo, increased levels of O-GlcNAc impair estrogen receptor activation and cause a decrease in mammary ductal side-branching morphogenesis associated with loss of progesterone receptors. Increased O-GlcNAc levels suppress transcriptional expression of coactivators and of the nuclear hormone receptors themselves. Surprisingly, increased O-GlcNAc levels are also associated with increased transcription of genes encoding corepressor proteins NCoR and SMRT. The association of the enzyme O-GlcNAc transferase with these corepressors contributes to specific regulation of nuclear hormone receptors by O-GlcNAc. Overall, transcriptional inhibition is related to the integrated effect of O-GlcNAc by direct modification of critical elements of the transcriptome and indirectly through O-GlcNAc modification of the proteasome.
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PMID:O-GlcNAc integrates the proteasome and transcriptome to regulate nuclear hormone receptors. 1696 74


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