Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.26.4 (RNase H)
 2,751 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Previous work from our laboratory demonstrated that CCAAT/enhancer-binding protein delta (C/EBP delta) functions in the initiation and maintenance of G(0) growth arrest in mouse mammary epithelial cells (MECs). In this report, we investigated the posttranscriptional and posttranslational regulation of C/EBP delta in G(0) growth-arrested mouse MECs. The results of transcriptional inhibitor studies demonstrated that the C/EBP delta mRNA exhibits a relatively short half-life in G(0) growth-arrested mouse MECs (t(1/2) approximately 35 min). In contrast, C/EBP delta mRNA has a longer half-life in G(0) growth-arrested mouse fibroblast cells (t(1/2) >100 min). Oligo/RNase H cleavage analysis and rapid amplification of cDNA ends-poly(A) test both confirmed the short C/EBP delta mRNA half-life observed in MECs and demonstrated that the C/EBP delta mRNA poly(A) tail is relatively short (approximately 100 nucleotides). In addition, the poly(A) tail length was not shortened during C/EBP delta mRNA degradation, which suggested a deadenylation-independent pathway. The C/EBP delta protein also exhibited a relatively short half-life in G(0) growth-arrested mouse MECs (t(1/2) approximately 120 min). The C/EBP delta protein was degraded in a ubiquitin-dependent manner, primarily in the nucleus, during G(0) growth arrest. In conclusion, these studies indicated that the C/EBP delta mRNA and protein content are under tight regulation in G(0) growth-arrested mouse MECs, despite the general concept that G(0) growth arrest is associated with a decrease in cellular activity.
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PMID:Posttranscriptional and posttranslational regulation of C/EBP delta in G0 growth-arrested mammary epithelial cells. 1255 32

We applied a combination of 15N relaxation and CSA/dipolar cross-correlation measurements at five magnetic fields (9.4, 11.7, 14.1, 16.4, and 18.8 T) to determine the 15N chemical shielding tensors for backbone amides in protein G in solution. The data were analyzed using various model-independent approaches and those based on Lipari-Szabo approximation, all of them yielding similar results. The results indicate a range of site-specific values of the anisotropy (CSA) and orientation of the 15N chemical shielding tensor, similar to those in ubiquitin (Fushman, et al. J. Am. Chem. Soc. 1998, 120, 10947; J. Am. Chem. Soc. 1999, 121, 8577). Assuming a Gaussian distribution of the 15N CSA values, the mean anisotropy is -173.9 to -177.2 ppm (for 1.02 A NH bond length) and the site-to-site CSA variability is +/-17.6 to +/-21.4 ppm, depending on the method used. This CSA variability is significantly larger than derived previously for ribonuclease H (Kroenke, et al. J. Am. Chem. Soc. 1999, 121, 10119) or recently, using "meta-analysis" for ubiquitin (Damberg, et al. J. Am. Chem. Soc. 2005, 127, 1995). Standard interpretation of 15N relaxation studies of backbone dynamics in proteins involves an a priori assumption of a uniform 15N CSA. We show that this assumption leads to a significant discrepancy between the order parameters obtained at different fields. Using the site-specific CSAs obtained from our study removes this discrepancy and allows simultaneous fit of relaxation data at all five fields to Lipari-Szabo spectral densities. These findings emphasize the necessity of taking into account the variability of 15N CSA for accurate analysis of protein dynamics from 15N relaxation measurements.
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PMID:Variability of the 15N chemical shielding tensors in the B3 domain of protein G from 15N relaxation measurements at several fields. Implications for backbone order parameters. 1677 99

The p53-Mdm2 system is key to tumor suppression. We have recently reported that p53 as well as Mdm2 are capable of supporting DNA replication fork progression. On the other hand, we found that Mdm2 is a modifier of chromatin, modulating polycomb repressor complex (PRC)-driven histone modifications. Here we show that, similar to Mdm2 knockdown, the depletion of PRC members impairs DNA synthesis, as determined in fiber assays. In particular, the ubiquitin ligase and PRC1 component RNF2/Ring1B is required to support DNA replication, similar to Mdm2. Moreover, the Ring finger domain of Mdm2 is not only essential for its ubiquitin ligase activity, but also for proper DNA replication. Strikingly, Mdm2 overexpression can rescue RNF2 depletion with regard to DNA replication fork progression, and vice versa, strongly suggesting that the two ubiquitin ligases perform overlapping functions in this context. H2A overexpression also rescues fork progression upon depletion of Mdm2 or RNF2, but only when the ubiquitination sites K118/K119 are present. Depleting the H2A deubiquitinating enzyme BAP1 reduces the fork rate, suggesting that both ubiquitination and deubiquitination of H2A are required to support fork progression. The depletion of Mdm2 elicits the accumulation of RNA/DNA hybrids, suggesting R-loop formation as a mechanism of impaired DNA replication. Accordingly, RNase H overexpression or the inhibition of the transcription elongation kinase CDK9 each rescues DNA replication upon depletion of Mdm2 or RNF2. Taken together, our results suggest that chromatin modification by Mdm2 and PRC1 ensures smooth DNA replication through the avoidance of R-loop formation.
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PMID:Chromatin modifiers Mdm2 and RNF2 prevent RNA:DNA hybrids that impair DNA replication. 3041 23

Proteins with a functionalized C-terminus such as a C-terminal thioester are key to the synthesis of larger proteins via expressed protein ligation. They are usually made by recombinant fusion to intein. Although powerful, the intein fusion approach suffers from premature hydrolysis and low compatibility with denatured conditions. To totally bypass the involvement of an enzyme for expressed protein ligation, here we showed that a cysteine in a recombinant protein was chemically activated by a small molecule cyanylating reagent at its N-side amide for undergoing nucleophilic acyl substitution with amines including a number of l- and d-amino acids and hydrazine. The afforded protein hydrazides could be used further for expressed protein ligation. We demonstrated the versatility of this activated cysteine-directed protein ligation (ACPL) approach with the successful synthesis of ubiquitin conjugates, ubiquitin-like protein conjugates, histone H2A with a C-terminal posttranslational modification, RNase H that actively hydrolyzed RNA, and exenatide that is a commercial therapeutic peptide. The technique, which is exceedingly simple but highly useful, expands to a great extent the synthetic capacity of protein chemistry and will therefore make a large avenue of new research possible.
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PMID:Expressed Protein Ligation without Intein. 3221 92