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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Assembly of hepadnaviruses depends on the formation of a ribonucleoprotein (RNP) complex comprising the viral polymerase polypeptide and an RNA segment, epsilon, present on pregenomic RNA. This interaction, in turn, activates the reverse transcription reaction, which is primed by a tyrosine residue on the polymerase. We have shown recently that the formation of this RNP complex in an avian hepadnavirus, the duck hepatitis B virus, depends on cellular factors that include the heat shock protein 90 (Hsp90). We now report that RNP formation also requires ATP hydrolysis and the function of p23, a recently identified chaperone partner for Hsp90. Furthermore, we also provide evidence that the chaperone complex is incorporated into the viral nucleocapsids in a polymerase-dependent reaction. Based on these findings, we propose a model for hepadnavirus assembly and priming of viral DNA synthesis where a dynamic, energy-driven process, mediated by a multi-component chaperone complex consisting of Hsp90, p23 and, potentially, additional factors, maintains the reverse transcriptase in a specific conformation that is competent for RNA packaging and protein priming of viral DNA synthesis.
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PMID:Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. 900 68

Reverse transcription in hepatitis B viruses is initiated through a unique protein priming mechanism whereby the viral reverse transcriptase (RT) first assembles into a ribonucleoprotein (RNP) complex with its RNA template and then initiates DNA synthesis de novo using the RT itself as a protein primer. RNP formation and protein priming require the assistance of host cell factors, including the molecular chaperone heat shock protein 90 (Hsp90). To better understand the mechanism of RT activation by Hsp90, we have now mapped the minimal RT sequences of the duck hepatitis B virus that are required for chaperone binding, RNP formation, and protein priming. Furthermore, we have reconstituted in vitro both RNP formation and protein priming using purified RT proteins and host factors. Our results show that (i) Hsp90 recognizes two independent domains of the RT, both of which are necessary for RNP formation and protein priming; (ii) Hsp90 function is required not only to establish, but also to maintain, the RT in a state competent for RNA binding; and (iii) Hsp90 is not required during RT synthesis and can activate the RT posttranslationally. Based on these findings, we propose a model for Hsp90 function whereby the chaperone acts as an active interdomain bridge to bring the two RT domains into a poised but labile conformation competent for RNP formation. It is anticipated that the reconstitution system established here will facilitate the isolation of additional host factors required for RT functions and further elucidation of the mechanisms of RT activation.
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PMID:In vitro reconstitution of a functional duck hepatitis B virus reverse transcriptase: posttranslational activation by Hsp90. 1109 Jan 40

The objective of the present study was to understand the role of cortisol in the cellular stress response process in fish. Specifically, our studies addressed whether cortisol exposure modified heat shock protein 90 (HSP90) mRNA expression in rainbow trout (Oncorhynchus mykiss) hepatocytes maintained in primary culture. We also subjected these hepatocytes to heat shock (HS) in order to examine the role of cortisol on HS-induced HSP90 mRNA expression. A cDNA fragment of 500 bp was cloned from trout liver by reverse transcriptase- polymerase chain reaction (RT-PCR) with primers designed from the conserved regions of chinook salmon and zebrafish HSP90 cDNAs. The PCR product showed very high homology to chinook salmon (98%), zebrafish (84%) and human (77%) HSP90. Heat shock (+6 degrees C) induced transient elevation in HSP90 mRNA in trout hepatocytes, peaking within 10-h post-HS, and remained elevated over a 24-h period. Cortisol did not modify the unstimulated expression of HSP90 mRNA, whereas the HS-induced HSP90 mRNA expression was attenuated in trout hepatocytes. Our results suggest that elevated plasma cortisol levels modulate the cellular stress response by affecting the transcription of HSP90 in fish.
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PMID:Cortisol modulates HSP90 mRNA expression in primary cultures of trout hepatocytes. 1139 5

Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase. Through down- or upregulation of telomerase, we found that only hTERT expression changed proportionally with the level of telomerase activity. The other components, TEP1, hTR, hsp90, p23, and dyskerin remained at high and unchanged levels throughout modulation. In vivo and in vitro experiments with antisense oligonucleotides against each telomerase component were also performed. Telomerase activity was decreased or abolished by antisense treatment. To correlate clinical sample status, four pairs of normal and malignant tissues from patients with oral cancer were examined. Except for the hTERT subunit, which showed differential expression in normal and cancer tissues, all other components were expressed in both normal and malignant tissues. We conclude that hTERT is a regulatable subunit, whereas the other components are expressed more constantly in cells. Although hTERT has a rate-limiting effect on enzyme activity, the other telomerase subunits (hTR, TEP1, hsp90, p23, dyskerin) participated in full enzyme activity. We hypothesize that once hTERT is expressed, all other telomerase subunits can be assembled to form a highly active holoenzyme.
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PMID:Differential regulation of telomerase activity by six telomerase subunits. 1213 83

The reverse transcriptase (RT) encoded by hepadnaviruses (hepatitis B viruses) is a multifunctional protein critical for several aspects of viral assembly and replication. Reverse transcription is triggered by the specific interaction between the RT and an RNA signal located on the viral pregenomic RNA, termed epsilon, and is initiated through a novel protein priming mechanism whereby the RT itself serves as a protein primer and epsilon serves as the obligatory template. Using the RT from duck hepatitis B virus as a model, we previously demonstrated that RT-epsilon interaction and protein priming require the assistance of a host cell chaperone complex, heat shock protein 90 (Hsp90) and its co-chaperones, which associates with the RT and facilitates the folding of the RT into an active conformation. We now report that extensive truncation removing the entire C-terminal RNase H domain and part of the central RT domain could relieve this dependence on Hsp90 for RT folding such that the truncated RT variants could function in epsilon interaction and protein priming independently of Hsp90. The presence of certain nonionic or zwitterionic detergent was sufficient to establish and maintain the truncated RT proteins in an active, albeit labile, state. Furthermore, we were able to refold an RT truncation variant de novo after complete denaturation. In contrast, the full-length RT and also RT variants with less-extensive C-terminal truncations required Hsp90 for activation. Surprisingly, the presence of detergent plus some yet-to-be-identified cytoplasmic factor(s) led to a dramatic suppression of the RT activities. These results have important implications for RT folding and conformational maturation, Hsp90 chaperone function, and potential inhibition of RT functions by host cell factors.
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PMID:Heat shock protein 90-independent activation of truncated hepadnavirus reverse transcriptase. 1266 54

The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-epsilon interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-epsilon interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific epsilon binding. We now report that Hsp90 is also required for HBV RT-epsilon interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-epsilon interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-epsilon binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-epsilon interaction in HBV, as in DHBV. Specific epsilon binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV epsilon, but not the DHBV epsilon, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of epsilon was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-epsilon interaction and chaperone activation.
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PMID:Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function. 1554 64

Initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA by the viral reverse transcriptase (RT). Using an in vitro reconstitution system whereby the cellular heat shock protein 90 chaperone system activates recombinant HBV RT for specific epsilon binding, we have defined the protein and RNA sequences required for specific HBV RT-epsilon interaction in vitro. Our results indicated that approximately 150 amino acid residues from the terminal protein domain and 230 from the RT domain were necessary and sufficient for epsilon binding. With respect to the epsilon RNA sequence, its internal bulge and, in particular, the first nucleotide (C) of the bulge were specifically required for RT binding. Sequences from the upper portion of the lower stem and the lower portion of the upper stem also contributed to RT binding, as did the base pairing of the upper portion and the single unpaired U residue of the upper stem. Surprisingly, the apical loop of epsilon, known to be required for RNA packaging, was entirely dispensable for RT binding. A comparison of the requirements for in vitro RT-epsilon interaction with those for in vivo pregenomic RNA (pgRNA) packaging clearly indicated that RT-epsilon interaction was necessary but not sufficient for pgRNA packaging. In addition, our results suggest that recognition of some epsilon sequences by the RT may be required specifically for viral DNA synthesis.
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PMID:Hepatitis B virus reverse transcriptase and epsilon RNA sequences required for specific interaction in vitro. 1647 22

The small DNA genome of hepadnaviruses is replicated by reverse transcription via an RNA intermediate. This RNA "pregenome" contains important signals that control critical steps of viral replication, including RNA packaging, initiation of reverse transcription, and elongation of minus strand DNA, through specific interactions with the viral reverse transcriptase, the capsid protein, and host factors. In particular, the interaction between the viral reverse transcriptase and RNA pregenome requires a host chaperone complex composed of the heat shock protein 90 and its cochaperones.
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PMID:RNA-protein interactions in hepadnavirus reverse transcription. 1927 50

Tolbutamide is used as a first line oral antihyperglycemic drug for type 2 diabetes. One side effect of this drug, hepatotoxicity, is well recognized; however, the precise mechanisms underlying tolbutamide-induced hepatotoxicity remain unclear. In this respect, proteomics techniques were used to gain further insight into the mechanistic processes of the hepatotoxicity induced by this drug. In this study, we aimed to identify molecular pathways based on proteins responding to cellular toxicity in tolbutamide-treated primary hepatocytes, using nano UPLC-MS/MS analysis. Rat primary hepatocytes were treated with an IC(20) concentration for 24 h to study the hepatotoxic effects of tolbutamide. For high-throughput label-free quantitation, tryptic-digested peptides of proteins from cell lysates were analyzed using LC-MS/MS and quantitated using the IDEAL-Q software, in which several parameters, such as assisted sequence, elution time, and mass-to-charge ratio were included. We quantified a total of 330 distinct proteins from the tolbutamide-treated hepatocytes and identified 55 upregulated and 82 downregulated proteins with expression changes. Among these differentially expressed proteins, we focused mainly on the 18 upregulated proteins belonging to xenobiotic cytochrome P450 (CYP), drug metabolism/detoxification, oxidative stress/antioxidant response, and cell damage pathway. CYP2D1, CYP2C11, UDP-glucuronosyltransferase 2B (UGT2B), superoxide dismutase 2 (SOD2), 60 kDa heat shock protein (HSPD1), heat shock protein 90 (HSP90), and catalase (CAT) were confirmed by Western blot analysis. In addition, various xenobiotic CYP proteins upregulated in the tolbutamide-treated group, CYP2D1, CYP2C13, and CYP2C11 were confirmed by reverse transcriptase-PCR analysis. Our results offer important new insights into the molecular mechanisms of tolbutamide-induced hepatotoxicity.
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PMID:Proteome profiling of tolbutamide-treated rat primary hepatocytes using nano LC-MS/MS and label-free protein quantitation. 2291 36

Ionizing radiation (IR) leads to fibrosing alveolitis (FA) after a lag period of several weeks to months. In a rat model, FA starts at 8 weeks after IR. Before that, at 5.5 weeks after IR, the transcription factors Sp1 (stimulating protein 1) and AP-1 (activator protein 1) are inactivated. To find genes/proteins that were down-regulated at that time, differentially expressed genes were identified in a subtractive cDNA library and verified by quantitative RT-PCR (reverse transcriptase polymerase chain reaction), western blotting and immunohistochemistry (IH). The mRNA of the molecular chaperone HSP90AB1 (heat shock protein 90 kDa alpha, class B member 1) was down-regulated 5.5 weeks after IR. Later, when FA manifested, HSP90ab1 protein was down-regulated by more than 90% in lung cells with the exception of mast cells. In most mast cells of the normal lung, both HSP90ab1 and HSP70, another major HSP, show a very low level of expression. HSP70 was massively up-regulated in all mast cells three months after irradiation whereas HSP90AB1 was up-regulated only in a portion of mast cells. The strong changes in the expression of central molecular chaperones may contribute to the well-known disturbance of cellular functions in radiation-damaged lung tissue.
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PMID:Down-regulation of heat shock protein HSP90ab1 in radiation-damaged lung cells other than mast cells. 2467 Jul 92


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