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 proteasome inhibitors, lactacystin and N-acetyl-leucyl-leucyl-norlucinal, caused a rapid and near-complete loss of approximately 22-23-kDa ubiquitinated nucleoproteins, which we have identified as monoubiquitinated nucleosomal histones H2A and H2B by immunological and two-dimensional electrophoretic techniques. In human SKBr3 breast tumor cells, depletion of monoubiquitinated histones by the proteasome inhibitors coincided with the accumulation of high molecular weight ubiquitinated proteins in both nucleoprotein and cytosolic fractions and decreased unconjugated ubiquitin in the cytosol, without changes in the nonubiquitinated core histones. Unconjugated ubiquitin was not detected in isolated tumor cell nuclei. A similar loss in monoubiquitinated histones occurred in cells harboring a defective, temperature-sensitive mutation of the ubiquitin-activating E1 enzyme, after these cells were elevated from 33 degrees C to the non-permissive temperature of 39 degrees C. DNA replication and RNA transcription were decreased by the proteasome inhibitors most strongly after 90% of the ubiquitin had been removed from ubiquitinated histones H2A and H2B, suggesting a relationship between the nucleosomal histone ubiquitin status and the processing of genetic information. Interestingly, although both proteasome inhibitors caused a generalized decrease in methionine incorporation into proteins, they strongly induced the synthesis of the hsp72 and hsp90 stress proteins. Finally, treating cells with heat-shock at 43 degrees C, with stress response-provoking chemicals or with several other proteasome inhibitors caused ubiquitinated proteins to accumulate, depleted free ubiquitin, and concomitantly decreased nucleosomal monoubiquitinated histones. These results suggest that deubiquitination of nucleosomal histones H2A and H2B may play a previously unrecognized role in the cellular stress response, as well as in the processing of chromatin, and emphasize the important role of the proteasome in cellular homeostasis.
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PMID:Rapid deubiquitination of nucleosomal histones in human tumor cells caused by proteasome inhibitors and stress response inducers: effects on replication, transcription, translation, and the cellular stress response. 939 60

Histones H2A and H2B are known to be reversibly post-translationally modified by ubiquitination. We previously observed in cultured tumor cells that proteasome inhibition stabilizes polyubiquitinated proteins, depletes unconjugated ubiquitin, and thereby promotes the deubiquitination of nucleosomal histones in chromatin. Provocative indirect evidence suggests that histone ubiquitination/deubiquitination cycles alter chromatin structure, which may limit accessibility of DNA repair proteins to damaged sites. In the present study, we focused on the relationship between the ubiquitination status of histone H2A, the structure of chromatin, and the efficiency of nucleotide excision repair (NER) of cisplatin-DNA adducts in human ovarian carcinoma cells exposed to the antitumor drug cisplatin. Pretreating cells with the proteasome inhibitor lactacystin (LC) or N-acetyl-leucyl-leucyl-norleucinal (ALLnL) induced deubiquitination of ubiquitinated histone H2A (uH2A) and concomitantly promoted chromatin condensation, increased the extent of cisplatin-DNA adducts, and diminished NER-dependent repair of cisplatin-DNA lesions, compared with control cells treated with cisplatin alone. Both proteasome inhibitors also prevented the increase in ERCC-1 mRNA expression that occurs in cells exposed to cisplatin. Cells treated with the combination of ALLnL and cisplatin underwent apoptosis, as indicated by caspase-dependent poly(ADP-ribose) polymerase (PARP) cleavage, more quickly than cells treated with either agent alone. Additionally, the combination of ALLnL and cisplatin potently increased p53 levels in cell lysates and stimulated the binding of p53 to chromatin. Together, these observations suggest that proteasome inhibition may be exploited therapeutically for its potential to sensitize ovarian tumor cells to cisplatin.
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PMID:Prevention of cisplatin-DNA adduct repair and potentiation of cisplatin-induced apoptosis in ovarian carcinoma cells by proteasome inhibitors. 1100 28

Small molecules suppressing proteasome function inhibit the post-translational ubiquitination of selected proteins. Ubiquitin H2A is an example of an abundant chromatin-associated protein that is known to be ubiquitinated, which is important for several proteins involved in the repair of DNA damage. We therefore studied the effect of the proteasome inhibitor, N-acetyl leucyl-leucyl norlucinal (ALLnL), on cisplatin sensitivity in three human ovarian tumor cell lines. The proteasome inhibitor ALLnL was administered for 4 h before cells were subsequently exposed to cisplatin for 1 h. Our results showed that ALLnL, at its respective IC20 concentration, increased cellular sensitivity to cisplatin in an additive manner in human ovarian cancer A2780, A2780/CP70, and OVCAR3 cells. We also demonstrated that ALLnL caused a 50% increase in total cellular accumulation of cisplatin, and reduced the rate of cisplatin efflux by about 50%. In addition, DNA damage levels were increased after ALLnL treatment. By contrast, DNA repair was inhibited 2 to 3-fold in ALLnL-pretreated cells, as compared to the controls. Furthermore, our study showed that ALLnL deubiquitinated nucleosomal histone H2A in these cells in a concentration-dependent fashion, as assessed by Western blot analysis. These data suggest that sublethal levels of exposure to agents that inhibit proteasome function may alter the subcellular pharmacology of platinum in human ovarian carcinoma cells.
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PMID:Effect of the proteasome inhibitor ALLnL on cisplatin sensitivity in human ovarian tumor cells. 1156 49

Although proteasomes are abundant in the nucleoplasm little is known of proteasome-dependent proteolysis within the nucleus. Thus, we monitored the subcellular distribution of nuclear proteins in correlation with proteasomes. The proteasomal pathway clears away endogenous proteins, regulates numerous cellular processes, and delivers immunocompetent peptides to the antigen presenting machinery. Confocal laser scanning microscopy revealed that histones, splicing factor SC35, spliceosomal components, such as U1-70k or SmB/B('), and PML partially colocalize with 20S proteasomes in nucleoplasmic substructures, whereas the centromeric and nucleolar proteins topoisomerase I, fibrillarin, and UBF did not overlap with proteasomes. The specific inhibition of proteasomal processing with lactacystin induced accumulation of histone protein H2A, SC35, spliceosomal components, and PML, suggesting that these proteins are normally degraded by proteasomes. In contrast, concentrations of centromeric proteins CENP-B and -C and nucleolar proteins remained constant during inhibition of proteasomes. Quantification of fluorescence intensities corroborated that nuclear proteins which colocalize with proteasomes are degraded by proteasome-dependent proteolysis within the nucleoplasm. These data provide evidence that the proteasome proteolytic pathway is involved in processing of nuclear components, and thus may play an important role in the regulation of nuclear structure and function.
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PMID:Proteasome-dependent processing of nuclear proteins is correlated with their subnuclear localization. 1249 Jan 67

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knock-down with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2alpha) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2alpha-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2alpha also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2alpha directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2alpha-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2alpha-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2alpha. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2alpha. We also propose that Sir2alpha is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasome-dependent pathway.
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PMID:Histone H2A.z is essential for cardiac myocyte hypertrophy but opposed by silent information regulator 2alpha. 1668 93

The reiterated nature of histone genes has hampered genetic approach to dissect the role of histones in chromatin dynamics. We here report isolation of three temperature-sensitive (ts) Schizosaccharomyces pombe strains, containing amino-acid substitutions in the sole histone H2B gene (htb1+). The mutation sites reside in the highly conserved, non-helical residues of H2B, which are implicated in DNA-protein or protein-protein interactions in the nucleosome. In the allele of htb1-72, the substitution (G52D) occurs at the DNA binding loop L1, causing disruption of the gene silencing in heterochromatic regions and lagging chromosomes in anaphase. In another allele htb1-223 (P102L) locating in the junction between alpha3 and alphaC, the mutant residue is in contact with H2A and other histones, leading to structural aberrations in the central centromere chromatin and unequal chromosome segregation in anaphase. The third allele htb1-442 (E34K) near alpha1 displayed little defect. Evidence is provided that monoubiquitinated H2B is greatly unstable in P102L mutant, possibly owing to proteasome-independent destruction or enhanced deubiquitination. Histone H2B thus plays an important role in centromere/kinetochore formation.
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PMID:Histone H2B mutations in inner region affect ubiquitination, centromere function, silencing and chromosome segregation. 1668 22

Histones are key components of chromatin. We investigated histone H2A-immunoreactive proteins in acute monocytic leukemia THP-1 cells using three polyclonal antibodies raised against peptides corresponding to distinct regions of H2A. Two unknown immunoreactive proteins (9- and 12-kDa proteins), H2A (14kDa) and ubiquitinated H2A (23kDa) were found in the cell lysates prepared by immediate direct addition of SDS-PAGE sample buffer to the cells as well as in the nuclear and chromatin fractions. However, they were not found in the cytoplasmic fraction. The unknown proteins were successfully purified by immunoaffinity chromatography from the cell nucleus extract and identified as 9-kDa H2A(1-87) and 12-kDa H2A(1-114), suggesting that both were produced by limited proteolysis of intact H2A(1-129). The truncated forms of H2A probably persisted as chromatin constituents, since the stability of H2A(1-87) in the chromatin fraction was sensitive to treatment with micrococcal nuclease, and H2A(1-114) was solubilized with lower ionic strength from the chromatin fraction obtained by micrococcal nuclease treatment. Truncated H2A proteins in THP-1 cells were transiently increased in amount by short-term treatment with phorbol 12-myristate 13-acetate or all-trans-retinoic acid, both of which induce macrophage-like differentiation. Furthermore, these increases were suppressed by preceding treatment with carbobenzoxy-l-leucyl-l-leucyl-l-leucinal (MG132) but not with carbobenzoxy-l-isoleucyl-gamma-t-butyl-l-glutamyl-l-alanyl-l-leucinal (PSI), both of which are generally known as proteasome inhibitors. Our results suggest that histone H2A is cleaved at least at two sites by protease(s) that remain obscure, and might affect chromatins in the early stage of THP-1 cell differentiation.
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PMID:Purification and characterization of C-terminal truncated forms of histone H2A in monocytic THP-1 cells. 1697 71

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.
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PMID:FACT and the proteasome promote promoter chromatin disassembly and transcriptional initiation. 1957 30

Sirt1 appear to be NAD(+)-dependent deacetylase that deacetylates histones and several non-histone proteins. In this study, we identified Sirt1 as a physical interaction partner of Tip60, which is a mammalian MYST-type histone acetyl-transferase that specifically acetylates histones H2A and H4. Although Tip60 also acetylates DNA damage-specific histone H2A variant H2AX in response to DNA damage, which is a process required for appropriate DNA damage response, overexpression of Sirt1 represses Tip60-mediated acetylation of H2AX. Furthermore, Sirt1 depletion by RNAi causes excessive acetylation of H2AX, and enhances accumulation of gamma-ray irradiation-induced MDC1, BRCA1, and Rad51 foci in nuclei. These findings suggest that Sirt1 functions as negative regulator of Tip60-mediated acetylation of H2AX. Moreover, Sirt1 deacetylates an acetylated Tip60 in response to DNA damage and stimulates proteasome-dependent Tip60 degradation in vivo, suggesting that Sirt1 negatively regulates the protein level of Tip60 in vivo. Sirt1 may thus repress excessive activation of the DNA damage response and Rad51-homologous recombination repair by suppressing the function of Tip60.
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PMID:Sirt1 physically interacts with Tip60 and negatively regulates Tip60-mediated acetylation of H2AX. 1989 90

The helix-loop-helix inhibitor of differentiation and DNA binding (Id1) is well known as an oncogene in various tumors. Although it has been reported that Id1 promotes several oncogenic processes, it is still unclear whether Id1 functions through epigenetic transcriptional regulation. In this study, we examined the effect of Id1 on polycomb group (PcG) proteins, which are crucial epigenetic gene silencers, and found that Id1 regulated the expression of Mel-18 and Bmi-1, both of which belong to polycomb repressive complex 1. We also confirmed that Id1 induced Mel-18 downregulation, which was mediated by the Akt pathway, and consequently upregulated the transcription of its target gene, c-Myc. Using a promoter-reporter, we demonstrated that Id1 regulated Bmi-1 transcription through c-Myc binding to its E-box in the promoter. Finally, we examined the activity of E3 ligase RING1b, whose catalytic activity is increased by binding with the RING finger protein Bmi-1, and found that Id1 overexpression enhanced RING1b E3 ligase activity leading to accumulation of H2A ubiquitination and ubiquitin/proteasome-mediated degradation of geminin. Taken together, our study provided a novel link between Id1 and PcG proteins and suggested that Id1 may contribute to tumor development through PcG-mediated epigenetic regulation.
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PMID:Id1 enhances RING1b E3 ubiquitin ligase activity through the Mel-18/Bmi-1 polycomb group complex. 2069 53


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