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)

The heat shock protein Hsp90 is known as an essential component of several signal transduction pathways and has now been identified as an essential host factor for hepatitis B virus replication. Hsp90 interacts with the viral reverse transcriptase to facilitate the formation of a ribonucleoprotein (RNP) complex between the polymerase and an RNA ligand. This RNP complex is required early in replication for viral assembly and initiation of DNA synthesis through a protein-priming mechanism. These results thus invoke a role for the Hsp90 pathway in the formation of an RNP.
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PMID:Hsp90 is required for the activity of a hepatitis B virus reverse transcriptase. 857 14

Most normal human diploid cells have no detectable telomerase; however, expression of the catalytic subunit of telomerase is sufficient to induce telomerase activity and, in many cases, will bypass normal senescence. We and others have previously demonstrated in vitro assembly of active telomerase by combining the purified RNA component with the reverse transcriptase catalytic component synthesized in rabbit reticulocyte extract. Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase. To our knowledge, p23 and Hsp90 are the first telomerase-associated proteins demonstrated to contribute to telomerase activity.
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PMID:Functional requirement of p23 and Hsp90 in telomerase complexes. 1019 82

Telomerase is a specialized reverse transcriptase that catalyzes elongation of the telomeric tandem repeat, TTAGGG, by addition of this sequence to the ends of existing telomeres. Human telomerase reverse transcriptase (hTERT) has been identified as a catalytic enzyme involved in telomere elongation that requires telomerase RNA, human telomerase RNA component (hTR), as an RNA template. We established a new method to express and purify soluble insect-expressed recombinant hTERT. The partially purified FLAG-hTERT retained the catalytic activity of telomerase in a complementation assay in vitro to exhibit telomerase activity in telomerase-negative TIG3 cell extract and in a reconstitution assay with FLAG-hTERT and purified hTR in vitro. FLAG-hTERT (D712A) with a mutation in the VDV motif exhibited no telomerase activity, confirming the authentic catalytic activity of FLAG-hTERT. The reconstituted complex of FLAG-hTERT and hTR in vitro was detected by electrophoretic mobility shift assay, and its activity was stimulated by more than 30-fold by TIG3 cell extract. This suggested that some cellular component(s) in the extract facilitated the reconstituted telomerase activity in vitro. Geldanamycin had no effect on the reconstituted activity but partially reduced the stimulated activity of the reconstituted telomerase by the TIG3 cell extract, suggesting that Hsp90 may contribute to the stimulatory effect of the cellular components.
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PMID:Telomerase activity reconstituted in vitro with purified human telomerase reverse transcriptase and human telomerase RNA component. 1081 33

In the present study we examined the localization and overexpression of heat shock proteins (hsps), mainly hsp90, in pancreatic carcinoma tissue compared with control tissue (including chronic pancreatitis and normal pancreas tissue), with the aid of immunohistochemical staining, in situ hybridization and reverse transcriptase polymerase chain reaction. Hsp90 alpha mRNA was overexpressed more highly in pancreatic carcinoma than in the control tissue. The proliferating-cell-nuclear-antigen labeling index was also high in pancreatic carcinoma tissue compared with the other tissue. These findings suggest that the overexpression of hsp90 alpha mRNA in carcinomas may be correlated with cell proliferation. However, hsp90 beta was constitutively overexpressed almost equally in all groups of pancreatic tissue including pancreatic carcinoma, chronic pancreatitis and normal pancreas tissue. Immunohistochemical staining demonstrated a differentiation in the expression of hsp90 between histological types of pancreatic carcinoma. These findings suggest that hsp90 alpha is involved in carcinogenesis and that hsp90 beta is correlated to structural conformation. Hsp90 alpha and hsp90 beta seem to perform different functions in tissue containing malignant cells. P53, MDM2 and WAF1, that were cell-cycle-related oncogene product were more strongly expressed in the nuclei of the cancer cells of the cancer tissue. Especially, MDM2 was more strongly expressed in mucinous carcinoma and the mucin secreting tissues surrounding pancreatic carcinoma tissue. The expression of MDM2 protein might also be correlated to secretion systems during structural conformation and be correlated to hsp90 beta.
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PMID:Overexpression and localization of heat shock proteins mRNA in pancreatic carcinoma. 1085 51

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (epsilon) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by epsilon and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of epsilon-dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.
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PMID:Reconstitution of a functional duck hepatitis B virus replication initiation complex from separate reverse transcriptase domains expressed in Escherichia coli. 1146 13

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
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PMID:In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins. 1173 92

The cellular chaperone Hsp90 has been shown to associate with the reverse transcriptase (RT) of the duck hepatitis B virus and is required for RT functions. However, the molecular basis for the specific interaction between the RT and Hsp90 remains unknown. Comparison of protein compositional properties suggests that the RT is highly related to the protein kinase c-Raf, which interacts with Hsp90 via the cochaperone p50 (CDC37). We tested whether the RT, like c-Raf, is specifically recognized by p50. Immunoprecipitation and pull-down assays showed that p50 or p50deltaC, a p50 mutant defective in Hsp90 binding, could interact specifically with the RT both in vitro and in vivo, indicating that p50 can bind the RT independently of Hsp90. Furthermore, purified p50 and p50deltaC interacted directly with purified RT. The importance of p50-RT interaction for RT functions was underscored by 1) inhibition of protein-primed initiation of reverse transcription by p50deltaC in vitro and 2) stimulation of viral DNA replication and RNA packaging by p50 and their inhibition by p50deltaC in transfected cells. These results suggest that p50 can function as a cellular cofactor for the hepadnavirus RT by mediating the interaction between the RT and Hsp90.
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PMID:Role of p50/CDC37 in hepadnavirus assembly and replication. 1198 22

In a continuation of our earlier study on the involvement of HSP25 (now known as Hspb1) and HSP70 (now known as Hspa4) in the induction of an adaptive response, we examined the involvement of these proteins in the induction of the adaptive response using an animal model system. C57BL6 mice were irradiated with 5 cGy of gamma radiation three times in 1 week (for a total of 15 cGy), and a high challenge dose (6 Gy) was given on the day after the last low-dose irradiation. The survival time of the low-dose preirradiated mice was increased to 30%. The induction of apoptosis induced by 6 Gy was also reduced by this low-dose preirradiation regimen. To elucidate any link existing between the HSPs and the induction of the adaptive response, reverse transcriptase (RT)-polymerase chain reaction (PCR) analysis was performed using splenocytes. High-dose radiation up-regulated the expression of Hspb1 and especially Hspa4, while expression of other HSPs such as HSC70 (now know as Hspa8), Hsp90, and alphaB-crystalline (now known as Cryab) did not change. When splenocytes from Hspa4 transgenic mice were preirradiated with a low dose of radiation, a reduction in cell death after high-dose irradiation was observed. These results suggest that Hspa4 is a key molecule in the induction of the adaptive response.
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PMID:Hspa4 (HSP70) is involved in the radioadaptive response: results from mouse splenocytes. 1200 43

Hsp90 is a specialized chaperone that controls the activity of many key regulator proteins such as steroid hormone receptors (SHRs). Hormone binding, and therefore SHR activation, requires Hsp90, which is loaded onto the receptors by a series of events involving Hsp70, Hsp40, Hop, and p23. The reverse transcriptase (RT) of hepatitis B viruses, small DNA-containing viruses that replicate via an RNA intermediate, has been reported to depend similarly on Hsp90 for enzymatic activity. Using an in vitro reconstitution system consisting of recombinant duck hepatitis B virus RT, purified chaperones, and the authentic RNA template Depsilon, we demonstrate here that this RT can be activated efficiently by just Hsp40 and Hsc70 plus energy, without the need for Hsp90 or other cofactors. The reaction appears to proceed selectively with the Hdj1 variant of Hsp40 but not Hdj2 or its yeast homolog Ydj1. The primary reaction product is a metastable, RNA binding-competent intermediate that decays quickly in the absence of its cognate RNA but, in its presence, accumulates in an initiation-competent form over several hours. Because deletion of the RNase H domain rendered the protein partly chaperone-independent, the chaperones may be needed indirectly to relieve occlusion of the RNA binding site by this domain. Our results do not exclude that other factors contribute to RT activation in vivo, but they challenge a fundamental SHR-like dependence on Hsp90. Thus Hsc70, mostly known for its role in general protein folding, is able to effect activation of a highly specialized target protein.
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PMID:Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein. 1285 1

We previously reported that gonocytes from 3-day-old rat testes proliferate in response to estradiol. In the present study, we found that purified gonocytes contained the mRNAs of estrogen receptor beta (ERbeta) and the chaperones Hsp90, p23, and Cyp40, but no inducible Hsp70. Immunoblot analysis showed high levels of ERbeta, Hsp90, p23, Cyp40, and the constitutive Hsc70 in gonocytes. Prenatal exposure to the estrogenic compounds diethylstilbestrol, bisphenol A, genistein, and coumestrol led to significantly increased Hsp90 mRNA levels in testis, but not p23 and Cyp40. In situ hybridization analysis indicated that Hsp90 mRNA was prominent in gonocytes, where it was increased following phytoestrogen exposure, whereas bisphenol A induced a more generalized increase throughout the testis. Immunoblot analysis of testicular extracts demonstrated that Hsp90 protein levels were significantly increased following estrogen exposure, and immunohistochemical analysis indicated that this increase occurred predominantly in gonocytes. By contrast, no change was observed in the expression of Cyp40, p23, and ERbeta, whereas Hsc70 was increased by bisphenol A only. Using an antibody and reverse transcriptase-polymerase chain reaction probes specific for Hsp90alpha, we subsequently confirmed that Hsp90alpha was primarily expressed in gonocytes, and that it was increased following estrogen exposure. Hsp90 immunolocalization in fetal and prepubertal testes showed an increased expression in fetal gonocytes upon estrogen exposure, but no difference in the subsets of Hsp90-positive germ cells in prepubertal testes. These results demonstrate that prenatal estrogen exposure specifically affects Hsp90 expression in gonocytes. Considering the interaction of Hsp90 with several signaling molecules, changes in its expression levels may lead to subsequent changes in gonocyte development.
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PMID:Prenatal estrogen exposure differentially affects estrogen receptor-associated proteins in rat testis gonocytes. 1522 38


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