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)

Automatic cell cycle analysis (DNA histograms) is usually performed with flow cytometry. In cases when only few cells are available, the DNA content has to be measured with a fluorescence microscope combined with sensitive camera systems (SIT, MCP, cooled CCDs) and a frame grabber for image analysis. The fluorescent cells are observed on the monitor of the image analyser. The DNA content of specific cells of interest is calculated after an interactive selection via mouse click on the monitor screen. It is desirable to automate this time-consuming procedure by a computer "cell-finding' and "cell-measurement' system, which is comparable in speed and measurement accuracy with manual scoring. A software program, based on image analysis, was designed for the automated cell finding, the cell measurement, and finally the interactive DNA data evaluation. The system has already been used to determine cell cycle stages in which specific manipulations of a lymphocyte culture were required. However, the system is also able to determine, automatically, DNA distributions of different kinds of cell (different leucocytes) within one sample, as long as the automatic recognition software can distinguish between them by morphological differences.
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PMID:Automated cell cycle analysis with fluorescence microscopy and image analysis. 877 30

HeLa cells were treated with different concentrations of an inhibitor of the proteasome chymotrypsin-like activity, the peptidyl aldehyde N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI). A detailed analysis, which included flow cytometry, cell counting and morphological assessment, was performed. PSI treatment induces a significant reduction of mitotic activity, accompanied by metaphase arrest of the mitotic cells. DNA flow cytometry shows an accumulation of the cells in G2+M phases of the cell cycle, which indicates the existence of a proteasome-mediated step in the G2-phase of the cell cycle. After removal of the inhibitor and supplementation with fresh medium, the cell cycle is resumed, but the mitotic cells show increased misalignment of chromosomes in the metaphase plate. PSI also induces HeLa cells to acquire a fibroblastoid phenotype.
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PMID:An inhibitor of the chymotrypsin-like activity of the multicatalytic proteinase complex (20S proteasome) induces arrest in G2-phase and metaphase in HeLa cells. 879 90

The human T-cell leukemia virus type I (HTLV-I) has been etiologically associated with the development of the adult T-cell leukemia (ATL) as well as degenerative neurologic syndrome termed tropical spastic paraparesis (TSP). HTLV-I encodes a potent transactivator protein termed Tax that appears to play an important role in the process of T-cell immortalization. Even though the mechanisms by which Tax induces transformation are still unknown, it seems likely that the ability of Tax to alter the expression of many cellular genes plays an important part in this process. Tax does not bind directly to DNA but rather deregulates the activity of cellular transcription factors. One family of host transcription factors whose activity is altered by Tax includes NF-kappa B/Rel. These transcription factors are post-transcriptionally regulated by their assembly with a second family of inhibitory proteins termed I kappa B that serve to sequester the NF-kappa B/Rel complexes in the cytoplasm. Upon cellular activation, I kappa B alpha is phosphorylated, polyubiquitinated, and degraded in the proteasome. This proteolytic event liberates NF-kappa B, permitting its rapid translocation into the nucleus where it binds to its cognate enhancer elements. Similarly, the p105 precursor of the NF-kappa B p50 subunit is also post-translationally processed in the proteasome. The mechanisms by which Tax activates NF-kappa B remain unclear, and findings presented in the literature are often controversial. We identified a physical interaction between Tax and the HsN3 subunit of the human proteasome. This raises the intriguing possibility that physical association of the HsN3 proteasome subunit with HTLV-I Tax coupled with the independent interaction of Tax with either p100 or p65-I kappa B alpha targets these cytoplasmic NF-kappa B/Rel complexes to the proteasome for processing.
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PMID:Interaction of HTLV-I Tax with the human proteasome: implications for NF-kappa B induction. 879 8

Recent work suggests that the proteolytic degradation of the nuclear lamins is a common event in apoptosis, although the nature of the proteases involved is still not clear. Our previous work showed that the degradation of lamin B1 in glucocorticoid-treated thymocytes occurs via a Ca2+-sensitive mechanism and that exogenous Ca2+ promotes lamin degradation in isolated thymocyte nuclei from untreated cells. Here we demonstrate that peptide-based inhibitors of the interleukin 1beta-converting enzyme family of cysteine proteases (Tyr-Val-Ala-Asp fluoromethyl ketone) and of the nuclear scaffold multicatalytic proteinase (Ala-Pro-Phe chloromethyl ketone) block the degradation of lamin B1 to a 21-kDa fragment in thymocytes treated with glucocorticoid, the Ca2+-mobilizing agent thapsigargin, or antibodies to the T cell receptor. However, among a panel of inhibitors specific for several different proteases implicated in apoptosis, only tosylphenylalanyl chloromethyl ketone and the nuclear scaffold protease inhibitor block lamin degradation, histone H1 cleavage, and DNA fragmentation in isolated thymocyte nuclei incubated with Ca2+. Overexpression of human BCL-2 in nuclei by stable transfection resulted in an inhibition of Ca2+-stimulated lamin degradation and DNA fragmentation, suggesting that endogenous nuclear BCL-2 regulates activation of the nuclear scaffold protease. The results demonstrate the existence of an alternative pathway of lamin degradation and DNA fragmentation mediated by a resident Ca2+-stimulated nuclear protease that is not directly dependent upon activation of the interleukin 1beta-converting enzyme family of cell death regulators.
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PMID:Calcium-dependent, interleukin 1-converting enzyme inhibitor-insensitive degradation of lamin B1 and DNA fragmentation in isolated thymocyte nuclei. 879 2

The human epithermoid carcinoma-derived cell line MA1, established by introduction of the adenovirus E1A 12 S cDNA linked to the mouse mammary tumor virus long terminal repeat, elicits apoptosis after induction of E1A12S in response to dexamethasone. The level of topoisomerase IIalpha begins to decrease steeply within 36 h preceding the onset of DNA fragmentation, whereas its mRNA level is unchanged (Nakajima, T., Ohi, N., Arai, T., Nozaki, N., Kikuchi, A., and Oda, K. (1995) Oncogene 10, 651-662). Topoisomerase IIalpha prepared by immunoprecipitation or extraction of the nuclear matrix was degraded much more efficiently in the S10 extract prepared from MA1 cells treated with dexamethasone for 42 h (the 42-h extract) than in the extract from untreated MA1 cells (the 0-h extract) in an ATP- and ubiquitin-dependent manner. The proteolytic activity for degradation of topoisomerase IIalpha was suppressed specifically by inhibitors for the proteasome and was much reduced in the 42-h extract prepared from MA1-derivative cell lines expressing E1B19k or Bcl-2. The proteolytic activity was lost after fractionation of the 42-h S10 extract into the S70 and P70 fractions by centrifugation at 70,000 x g for 6 h but partially recovered when these fractions were combined. Polyubiquitinated forms of topoisomerase IIalpha could be detected by incubating it in the S70 or S100 extract, which lacks most of the proteasome activity. The ubiquitination activity in S70 prepared from the 42-h extract was 4- to 5-fold higher than that prepared from the 0-h extract. These results suggest that a component(s) in the ubiquitin proteolysis pathway, responsible for ubiquitination and degradation of topoisomerase IIalpha, is activated or induced during the latent phase of E1A-induced apoptosis.
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PMID:Degradation of topoisomerase IIalpha during adenovirus E1A-induced apoptosis is mediated by the activation of the ubiquitin proteolysis system. 879 59

We have cloned a porcine gene, designated TBP1O, that belongs to the Tat-binding protein/26S protease subunit family. The genomic structure of the porcine TBP1O gene was analyzed after isolation of three overlapping genomic phage lambda clones. The TBP10 gene harbors 12 exons spanning 4.5 kb of chromosomal DNA. The TBP1O gene was assigned to Chromosome (Chr) 12 by fluorescence in situ hybridization (FISH) on metaphase chromosomes. The chromosomal location was confirmed by PCR analysis of a porcine-rodent hybrid cell panel. The TBP1O protein is encoded by a 1221 nucleotide cDNA and has a molecular mass of 45.6 kDa. The predicted amino acid sequence has highest similarity to the human and bovine p45 subunit of the 26S protease and the human transcription factor TRIP1. Further similarities were detected to the slime mold protein DdTBP1O and the Schizosaccharomyces pombe and Saccharomyces cerevisiae protein SUG1. Like DdTBP1O and other members of the protein family, the porcine TBP1O harbors a leucine zipper motif in the N-terminal region and a domain characteristics of ATP-dependent proteases in the C-terminal region.
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PMID:The porcine gene TBP10 encodes a protein homologous to the human tat-binding protein/26S protease subunit family. 883 36

Rb protein is a critical regulator of entry into the cell cycle, and loss of Rb function by deletions, mutations, or interaction with DNA viral oncoproteins leads to oncogenic transformation. We have shown that the human papilloma virus (HPV)-16 E7 gene is sufficient to induce the immortalization of mammary epithelial cells (MECs). Surprisingly, the steady-state level of Rb protein in these immortal cells was drastically decreased. Here, we used pulse-chase analysis to show that the in vivo loss of Rb protein in E7-immortalized MECs is a consequence of enhanced degradation. Expression of HPV16 E7 in a cell line with a temperature-sensitive mutation in the E1 enzyme of the ubiquitin pathway demonstrated that degradation of Rb was ubiquitin dependent. Treatment of E7-immortalized MECs with aldehyde inhibitors of proteasome-associated proteases led to a marked stabilization of Rb protein, particularly the hypophosphorylated form. Taken together, our results provide evidence for HPV-16 E7-induced enhanced degradation of Rb protein via a ubiquitin-proteasome pathway and suggest a second mechanism of oncogenic transformation by E7, in addition to its previously identified ability to sequester Rb from E2F. Our analyses also show that normal Rb levels are regulated by the ubiquitin-proteasome degradation pathway.
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PMID:E7 protein of human papilloma virus-16 induces degradation of retinoblastoma protein through the ubiquitin-proteasome pathway. 884 Sep 74

Exotoxin A production in Pseudomonas aeruginosa is a complicated and highly regulated process that involves several genes. In this report, we describe the isolation of a new toxA regulatory gene (ptxR) which affects exotoxin A production in P. aeruginosa. In an iron-deficient medium, the presence of a plasmid carrying ptxR in P. aeruginosa PAO1 resulted in a four-to fivefold increase in exotoxin A synthesis. No effect was observed on the levels of elastase, phospholipase C, exoenzyme S, and alkaline protease. Using subcloning and complementation experiments, ptxR was localized to a 2.1 kb Kpnl-Bg/II fragment. Nucleotide sequence analysis revealed the presence of an open reading frame which encodes a 34.97 kDa protein (PtxR). The size of the predicted PtxR compares closely with the 34 kDa PtxR that was synthesized in Escherichia coli using the T7 expression system. The deduced amino acid sequence of PtxR is homologous to that of several members of the LysR family of transcriptional activators. The amino-terminus region of PtxR contains a putative helix-turn-helix DNA-binding motif. Specific ptxR-deletion mutants in P. aeruginosa strains PAO1 and PA103 were constructed. In comparison with their parent strains, both mutants showed a significant reduction in the level of exotoxin A activity. However, upon extensive subculturing, the level of exotoxin A produced by the PAO1::ptxR mutant was similar to that of PAO1. Transcriptional studies, using both toxA-lacZ fusion and RNA analysis, confirmed that ptxR increases toxA and regA transcription. These results suggest that ptxR regulates (through regA) exotoxin A production at the transcriptional level.
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PMID:Isolation and characterization of a Pseudomonas aeruginosa gene, ptxR, which positively regulates exotoxin A production. 884 37

Proteolytic degradation of the C-terminal region of NF-(kappa)B precursors to their active DNA binding forms represents an important regulatory step in the activation of NF-(kappa)B. NF-(kappa)B2(p100) is found ubiquitously in the cytoplasm; however, the site and mechanism of processing to p52 have not previously been defined. We show by deletion mapping that processing of NF-(kappa)B2(p100) terminates at alanine 405 to generate p52 and is prevented by specific inhibitors of the multicatalytic proteinase complex. Although the C-terminal I(kappa)B-like domain of NF-(kappa)B2(p100) was constitutively phosphorylated, disruption of this phosphorylation by mutagenesis demonstrated that it was not required as a signal to mediate processing. Mutational analysis further showed that cleavage of NF-(kappa)B2 is not dependent on a specific sequence motif adjacent to alanine 405, the ankyrin repeats, or other C-terminal sequences but is directed by structural determinants amino terminal to the cleavage site, within the Rel homology domain and/or the glycine hinge region. The level of processing of NF-(kappa)B2(p100) was much lower than that of NF-(kappa)B1(p105) and differed from that of I(kappa)B-alpha, suggesting differential control of processing of NF-(kappa)B/I(kappa)B family members.
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PMID:Differential regulation of NF-kappaB2(p100) processing and control by amino-terminal sequences. 888 65

RAD6 in the yeast Saccharomyces cerevisiae encodes a ubiquitin-conjugating enzyme essential for DNA repair as well as for a number of other biological processes. It is believed that the functions of Rad6p require the ubiquitination of target proteins, but its substrates as well as other interacting proteins are largely unknown. Rad6p homologues of higher eukaryotes have a number of amino acid residues in the C-terminal alpha-helix, which are conserved from yeast to man but are absent from most other yeast ubiquitin-conjugating enzymes (Ubcs). This specific conservation suggests that the C-terminal alpha-helix is important for the unique activities of the Rad6p family of Ubcs. We have investigated the effects of mutating this highly conserved region on the ubiquitination of model substrates in vitro and on error-free DNA repair in vivo. C-terminal point and deletion mutants of Rad6p differentially affected its in vitro activity on various substrates, raising the possibility that Rad6p interacts with its substrates in vivo by similar mechanisms. The distal part of the C-terminal alpha-helix is also essential for error-free DNA repair in vivo. Overexpression of Rad18p, a single-stranded DNA-binding protein that also interacts with Rad6p, alleviates the DNA repair defects of the C-terminal alpha-helix mutants to different degrees. This indicates that the C-terminal alpha-helix of Rad6p mediates its interaction with Rad18p, an essential step in DNA repair. Models of Rad6p action propose that its ubiquitination function is followed by proteolysis of unknown ubiquitinated targets. Mutants affecting several functions of the 26S proteasome retain wild-type capacity for error-free DNA repair. This raises the possibility that ubiquitination by Rad6p in DNA repair does not target proteins for proteasomal degradation.
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PMID:Role of the conserved carboxy-terminal alpha-helix of Rad6p in ubiquitination and DNA repair. 889 88


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