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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

cDNA clone MS73 codes for an ATPase that is a regulatory subunit of the 26 S proteasome. Reverse transcriptase polymerase chain reaction analysis demonstrates that the expression of the gene dramatically increases in the pre-eclosion period. Western analyses show increases in other related. ATPases including MS73, MSS1, and mts2 but not TBP1. A similar increase in the 30-kDa subunit of the 20 S proteasome occurs. There are accompanying large changes in the peptidase activities of the 26 S proteasome. Relative to the 30-kDa subunit, there is no change in MSS1 and MS73, a 3-fold increase in mts2, and a 5-fold decline in TBP1. A large increase in the concentration of 26 S proteasomes together with extensive regulatory reprogramming may facilitate rapid muscular proteolysis.
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PMID:Developmental changes of the 26 S proteasome in abdominal intersegmental muscles of Manduca sexta during programmed cell death. 782 21

Epidemiologic studies in humans as well as immunohistologic studies in animals have demonstrated significant sex differences in the propensity to develop cataract. Several studies suggest that estrogen may play a protective role against cataractogenesis. Indeed, male and ovariectomized female rat lenses have a greater susceptibility to cataract induced by transforming growth factor-beta (TGF-beta) than do normal female lenses. However, in spite of the current evidence that estrogen may play a pivotal role in cataractogenesis, the molecular mechanisms behind this phenomenon are largely undetermined. Our study utilized the differential display procedure to examine gene up- and down-regulation in male, normal female and ovariectomized female rat lenses exposed to TGF-beta. Male and normal female rat lenses were cultured with or without 0.15 ng ml(-1)TGF-beta. Lenses were then harvested, and total RNA was isolated for analysis by reverse-transcriptase differential display. Differentially expressed mRNAs were subcloned, sequenced and identified through GenBank database searches. The original experiment was repeated with the addition of ovariectomized female TGF-beta(+/-) conditions, and all differential patterns of gene expression were verified using Northern blot and RT-PCR analysis. Screening of approximately 12% of the mRNA population led to the identification of 27 differentially expressed cDNAs. Notably, strong gender differences were found in expression levels of gammaB-crystallin. In addition, proteasome Z subunit was up-regulated in TGF-beta-treated male and ovariectomized female lenses, but was down-regulated in TGF-beta-treated normal female lenses. This pattern of expression is consistent with the increased susceptibility of male and ovariectomized lenses to TGF-beta-induced cataract. We conclude that differential display is a useful and expedient method for analysing changes in gene expression in the lens. Structural and functional studies of the genes identified in this study may further elucidate mechanisms underlying the TGF-beta-induced cataract formation and differential rates of cataractogenesis in males vs females. In particular, our data suggest that the role of proteasome Z subunit in TGF-beta-induced anterior subcapsular cataract warrants further investigation.
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PMID:Differential gene expression in male and female rat lenses undergoing cataract induction by transforming growth factor-beta (TGF-beta). 1065 42

All RNA viruses known to date encode an RNA-dependent RNA polymerase (RdRp) that is required for replication of the viral genome. We have expressed and purified the turnip yellow mosaic virus (TYMV) RdRp in insect cells using a recombinant baculovirus, either in its native form, or fused to an hexa-histidine tag. Phosphorylation of the protein was demonstrated by labelling experiments in vivo, as well as phosphatase treatment of the purified protein in vitro. Phospho amino acid analysis and immunoblotting experiments identified serine and threonine residues as being the subject of phosphorylation. Peptide mass mapping using MS analysis of a protein digest revealed that phosphorylation sites are localized within a putative PEST sequence [a sequence rich in proline (P), glutamic acid (E), serine (S) and threonine (T) residues] in the N-terminal region of the protein. Using monoclonal antibodies specific for ubiquitin conjugates, we were able to demonstrate that the TYMV RdRp is conjugated to ubiquitin molecules when expressed in insect cells. These observations suggest that the TYMV RdRp may be processed selectively by the ubiquitin/proteasome degradation system upon phosphorylation of the PEST sequence.
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PMID:Evidence for phosphorylation and ubiquitinylation of the turnip yellow mosaic virus RNA-dependent RNA polymerase domain expressed in a baculovirus-insect cell system. 1088 Mar 40

We demonstrated previously that nonsteroidal anti-inflammatory drugs (NSAIDs) increased p27(Kip1) by inhibiting protein degradation to suppress the proliferation of human lung cancer cells. In this study, we elucidate the molecular mechanism by which NSAIDs modulate p27(Kip1) proteolysis. Immunoblotting and in vitro ubiquitination assays indicated that the expression of Cul1 and Skp2 and ubiquitination activity toward p27(Kip1) were not regulated by NSAIDs. On the contrary, we found that NSAIDs inhibited proteasome activity to increase p27(Kip1) protein levels. NSAIDs suppressed the expression of chymotrypsin-like catalytic subunits (beta5, LMP7, and LMP2), but did not directly block enzymatic activity, to inhibit proteasome activity. Reverse transcriptase-competitive polymerase chain reaction and promoter activity assays showed that this inhibition occurred at the transcriptional level. In vitro degradation experiments showed that p27(Kip1) degradation was inhibited by NS398, and the addition of purified 26S proteasome reversed this inhibitory effect. Collectively, our results revealed the mechanism by which NSAIDs modulate p27(Kip1) protein degradation and suggest that NSAIDs are a novel class of proteasome inhibitors.
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PMID:Mechanisms underlying nonsteroidal anti-inflammatory drug-induced p27(Kip1) expression. 1243 20

The hepatitis A virus 3C protease and 3D RNA polymerase are present in low concentrations in infected cells. The 3C protease was previously shown to be rapidly degraded by the ubiquitin/26S proteasome system and we present evidence here that the 3D polymerase is also subject to ubiquitination-mediated proteolysis. Our results show that the sequence (32)LGVKDDWLLV(41) in the 3C protease serves as a protein destruction signal recognized by the ubiquitin-protein ligase E3alpha and that the destruction signal for the RNA polymerase does not require the carboxyl-terminal 137 amino acids. Both the viral 3ABCD polyprotein and the 3CD diprotein were also found to be substrates for ubiquitin-mediated proteolysis. Attempts to determine if the 3C protease or the 3D polymerase destruction signals trigger the ubiquitination and degradation of these precursors yielded evidence suggesting, but not unequivocally proving, that the recognition of the 3D polymerase by the ubiquitin system is responsible.
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PMID:Signals in hepatitis A virus P3 region proteins recognized by the ubiquitin-mediated proteolytic system. 1275 77

The ubiquitin-proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.
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PMID:Small ubiquitin-like modifying protein isopeptidase assay based on poliovirus RNA polymerase activity. 1635 62

The RNA-dependent RNA polymerase of influenza virus is a heterotrimer formed by the PB1, PB2, and PA subunits. Although PA is known to be required for polymerase activity, its precise role is still unclear. Here, we investigated the function of the N-terminal region of PA. Protease digestion of purified recombinant influenza virus A/PR/8/34 PA initially suggested that its N-terminal region is folded into a 25-kDa domain. We then systematically introduced point mutations into evolutionarily conserved amino acids in the N-terminal region of influenza virus A/WSN/33. Most alanine-scanning mutations between residues L109 and F117 caused PA degradation, mediated by a proteasome-ubiquitin pathway, and as a consequence interfered with polymerase activity. Three further PA mutations, K102A, D108A, and K134A, were investigated in detail. Mutation K102A caused a general decrease both in transcription and replication in vivo, whereas mutations D108A and K134A selectively inhibited transcription. Both the D108A and K134A mutations completely inhibited endonuclease activity in vitro, explaining their selective defect in transcription. K102A, on the other hand, resulted in a significant decrease in both cap binding and viral RNA promoter-binding activity and consequently inhibited both transcription and replication. These results suggest that the N-terminal region of PA is involved in multiple functions of the polymerase, including protein stability, endonuclease activity, cap binding, and promoter binding.
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PMID:Amino acid residues in the N-terminal region of the PA subunit of influenza A virus RNA polymerase play a critical role in protein stability, endonuclease activity, cap binding, and virion RNA promoter binding. 1687 36

The aminoacyl t-RNA synthetase interacting multifunctional protein (AIMP1) is the precursor of the multifunctional inflammatory cytokine endothelial monocyte-activating polypeptide II (EMAP II). We previously demonstrated that AIMP1 secretion by pituitary adenomas is inversely correlated with tumor diameter and with RARS expression, suggesting that a high amount of RARS associated with AIMP1 might prevent the secretion of the latter cytokine. In this study, we investigated the role of RARS in modulating the secretion of AIMP1 in HeLa and MCF7 cell lines and investigated the possible role of the multicatalytic protease in the cleavage of AIMP1 to generate EMAP II. Our data show that RARS over-expression impairs AIMP1 secretion by both HeLa and MCF7 cells. Moreover, proteasome inhibition impairs AIMP1 cleavage to produce EMAP II. These data indicate that RARS over-expression associates with a reduced AIMP1 secretion and that the multicatalytic protease is involved in the generation of the mature cytokine, EMAP II.
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PMID:Proteasomes and RARS modulate AIMP1/EMAP II secretion in human cancer cell lines. 1744 84

The molecular chaperone heat shock protein 90 (Hsp90) is involved in multiple cellular processes including protein maturation, complex assembly and disassembly, and intracellular transport. We have recently shown that a disruption of Hsp90 activity in cultured Drosophila melanogaster cells suppresses Flock House virus (FHV) replication and the accumulation of protein A, the FHV RNA-dependent RNA polymerase. In the present study, we investigated whether the defect in FHV RNA polymerase accumulation induced by Hsp90 suppression was secondary to an effect on protein A synthesis, degradation, or intracellular membrane association. Treatment with the Hsp90-specific inhibitor geldanamycin selectively reduced FHV RNA polymerase synthesis by 80% in Drosophila S2 cells stably transfected with an inducible protein A expression plasmid. The suppressive effect of geldanamycin on protein A synthesis was not attenuated by proteasome inhibition, nor was it sensitive to changes in either the mRNA untranslated regions or protein A intracellular membrane localization. Furthermore, geldanamycin did not promote premature protein A degradation, nor did it alter the extremely rapid kinetics of protein A membrane association. These results identify a novel role for Hsp90 in facilitating viral RNA polymerase synthesis in Drosophila cells and suggest that FHV subverts normal cellular pathways to assemble functional replication complexes.
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PMID:A functional heat shock protein 90 chaperone is essential for efficient flock house virus RNA polymerase synthesis in Drosophila cells. 1752 96

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infections and is uniquely associated with the development of hepatocellular carcinoma. While the mechanism(s) by which the virus promotes cancer are poorly defined, previous studies indicate that the HCV RNA-dependent RNA polymerase, nonstructural protein 5B (NS5B), forms a complex with the retinoblastoma tumor suppressor protein (pRb), targeting it for degradation, activating E2F-responsive promoters, and stimulating cellular proliferation. Here, we describe the mechanism underlying pRb regulation by HCV and its relevance to HCV infection. We show that the abundance of pRb is strongly downregulated, and its normal nuclear localization altered to include a major cytoplasmic component, following infection of cultured hepatoma cells with either genotype 1a or 2a HCV. We further demonstrate that this is due to NS5B-dependent ubiquitination of pRb and its subsequent degradation via the proteasome. The NS5B-dependent ubiquitination of pRb requires the ubiquitin ligase activity of E6-associated protein (E6AP), as pRb abundance was restored by siRNA knockdown of E6AP or overexpression of a dominant-negative E6AP mutant in cells containing HCV RNA replicons. E6AP also forms a complex with pRb in an NS5B-dependent manner. These findings suggest a novel mechanism for the regulation of pRb in which the HCV NS5B protein traps pRb in the cytoplasm, and subsequently recruits E6AP to this complex in a process that leads to the ubiquitination of pRb. The disruption of pRb/E2F regulatory pathways in cells infected with HCV is likely to promote hepatocellular proliferation and chromosomal instability, factors important for the development of liver cancer.
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PMID:Hepatitis C virus induces E6AP-dependent degradation of the retinoblastoma protein. 1790 5


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