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)

Muscle protein degradation is thought to play a major role in muscle atrophy in cancer cachexia. To investigate the importance of the ubiquitin-proteasome pathway, which has been suggested to be the main degradative pathway mediating progressive protein loss in cachexia, the expression of mRNA for proteasome subunits C2 and C5 as well as the ubiquitin-conjugating enzyme, E2(14k), has been determined in gastrocnemius and pectoral muscles of mice bearing the MAC16 adenocarcinoma, using competitive quantitative reverse transcriptase polymerase chain reaction. Protein levels of proteasome subunits and E2(14k) were determined by immunoblotting, to ensure changes in mRNA were reflected in changes in protein expression. Muscle weights correlated linearly with weight loss during the course of the study. There was a good correlation between expression of C2 and E2(14k) mRNA and protein levels in gastrocnemius muscle with increases of 6-8-fold for C2 and two-fold for E2(14k) between 12 and 20% weight loss, followed by a decrease in expression at weight losses of 25-27%, although loss of muscle protein continued. In contrast, expression of C5 mRNA only increased two-fold and was elevated similarly at all weight losses between 7.5 and 27%. Both proteasome functional activity, and proteasome-specific tyrosine release as a measure of total protein degradation was also maximal at 18-20% weight loss and decreased at higher weight loss. Proteasome expression in pectoral muscle followed a different pattern with increases in C2 and C5 and E2(14k) mRNA only being seen at weight losses above 17%, although muscle loss increased progressively with increasing weight loss. These results suggest that activation of the ubiquitin-proteasome pathway plays a major role in protein loss in gastrocnemius muscle, up to 20% weight loss, but that other factors such as depression in protein synthesis may play a more important role at higher weight loss.
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PMID:Expression of the ubiquitin-proteasome pathway and muscle loss in experimental cancer cachexia. 1616 Jun 95

Acquisition of drug-resistance conferring mutations leads to an enhanced degradation of HIV-1 reverse transcriptase (RT) affecting its immunogenicity. The mechanism of this degradation is not known. We investigated the input of proteasome in this degradation, and explored a possibility to enhance the proteasomal degradation of RTs to potentiate the immunogenic peformance of RT genes. To this end, a C-terminal fusion was made of RT with ornithine decarboxylase (ODC) that is rapidly degraded by proteasome in an ubiquitine-independent fashion. Eukaryotic cells were transiently transfected with the genes for wild-type (wt) RT, multi-drug-resistant (MDR) RT, and their chimeras with ODC. RT expression in the presence or absence of the proteasome inhibitors MG132 and epoxomicin was quantified by Western blotting. Treatment with MG132 led to a two-fold increase in the level of wtRT, and a four-fold increase in the level of MDR-RT accumulation. Treatment with epoxomicin had virtually no effect on the accumulation of wtRT, while stabilizing MDR-RT two-fold. Since epoxomicin is a more specific proteasome inhibitor, it indicated that degradation of wtRT may not be solely proteasomal. Fusion to ODC considerably decreased the intracellular levels of both RT-ODC and MDR-RT-ODC as compared to parental proteins. MG132 treatment increased the intracellular RT-ODC content 20-fold (up the level of the MG132-treated wtRT; 60-80 fg/cell), and epoxomicin treatment, 10-fold as compared to non-treated samples. Thus, attachment of ODC moiety has modified the metabolic pathway of RT targeting it to proteasomal degradation. We are currently testing if this is translated into an enhanced MHC class I performance of wild-type and drug-resistant RTs in gene immunization.
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PMID:HIV-1 reverse transcriptase targeted for proteasomal degradation as a prototype vaccine against drug-resistant HIV-1. 1618 8

Gentamicin accumulates in the lysosomes of kidney proximal tubular cells and causes apoptosis at clinically relevant doses. Gentamicin-induced apoptosis can be reproduced with cultured renal cells, but only at high extracellular concentrations (1 to 3 mM; 0.4 to 1.2 g/liter) because of its low level of uptake. We recently showed that gentamicin-induced apoptosis in LLC-PK1 cells involves a rapid (2-h) permeabilization of lysosomes and activation of the mitochondrial pathway of apoptosis (10 h). We now examine whether the delivery of gentamicin to the cytosol by electroporation would sensitize LLC-PK1 cells to apoptosis. Cells were subjected to eight pulses (1 ms) at 800 V/cm (square waves) in the presence of gentamicin (3 microM to 3 mM; 1.2 mg/liter to 1.2 g/liter); returned to gentamicin-free medium; and examined at 8 h for their Bax (a marker of mitochondrial pathway activation) contents by Western blotting and competitive reverse transcriptase PCR and at 24 h for apoptosis by 4',6'-diamidino-2'-phenylindole staining (confirmed by electron microscopy) and for necrosis (by determination of lactate dehydrogenase release). Nonelectroporated cells were incubated with gentamicin for 8 and 24 h. Significant increases in Bax levels (8 h) and apoptosis (24 h) were detected with 0.03 mM (13.2 mg/liter) gentamicin in electroporated cells compared with those achieved with 2 mM (928 mg/liter) in incubated cells. The increase in the Bax level was not associated with an increase in the level of its mRNA but was associated with the accumulation of ubiquitinated forms (probably as a result of impairment of its degradation by the proteasome). Assay of cell-associated gentamicin showed a marked, immediate, but transient accumulation in electroporated cells, whereas a slow, steady uptake was detected in incubated cells. The data indicate that cytosolic gentamicin triggers apoptosis. Sequestration of gentamicin in lysosomes would, to some extent, protect against apoptosis.
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PMID:Gentamicin causes apoptosis at low concentrations in renal LLC-PK1 cells subjected to electroporation. 1656 31

Telomerase counteracts loss of terminal sequences incurred during DNA replication. In S. cerevisiae, telomerase contains an RNA template (TLC1), a reverse transcriptase (Est2p) and at least two regulatory proteins (Est1p and Est3p). Whereas Est2p is constitutively telomere bound, Est1p associates in late S phase, coincident with telomere lengthening. Here we directly demonstrate by coimmunoprecipitation that the composition of telomerase varies during the cell cycle. The absence of Est1p and Est3p from the complex during G1 phase can be attributed to proteasome-dependent degradation of Est1p. Stabilization of Est1p during G1 phase promotes telomerase assembly, revealing a previously uncharacterized role for Est1p in the recruitment of Est3p to the telomerase complex. Though catalytically active, complexes assembled during G1 cannot lengthen telomeres. We conclude that telomerase assembly during G1 phase is regulated by Est1p stability, but assembly is insufficient to activate telomerase at telomeres.
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PMID:Proteasome-dependent degradation of Est1p regulates the cell cycle-restricted assembly of telomerase in Saccharomyces cerevisiae. 1686 58

Proteasome plays a key role in antigen presentation through MHC class I pathway. Thus, approaches are actively developed to increase proteasome targeting of DNA-vaccine encoded proteins. Gene of reverse transcriptase of HIV-1 is used in DNA-vaccines. It was shown, that revertase degraded in cells slowly (half-life is 18-20 h). Revertase content increased in presence of proteasome inhibitors MG132 and epoxomicin indicated that it degraded by proteasome. Level of protein was 2 fold higher after treatment with MG132 then after epoxomicin treatment. Since epoxomicin is more specific proteasome inhibitor it indicated that other cellular proteases can take part in revertase degradation. With the aim to increase affinity and degradation rate by proteasome of revertase we have to add strong degradation signal. Ornithine decarboxylase contains this kind of signals, it's unique properties are fast degradation by proteasome in ubiquitin-independent manner. As result fusion protein of revertase and ornithine decarboxylase was created. Half-life of fusion protein was 6 time less than revertase (3 h). Degradation of fusion protein was blocked by proteasome inhibitors 10 times stronger than revertase. Thus, degradation by proteasome pathway of reverse transcriptase was enhanced by fusion with ornithine decarboxylase. Performance of this fusion could improve presentation of revertase in DNA-vaccine.
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PMID:[Artifitial increase of HIV-1 reverse transcriptase turnover through proteasome pathway]. 1720 25

Targeting of a DNA vaccine encoded protein for degradation via the proteasome is attempted since it may enhance the immunogenicity of the vaccine. We have fused HIV-1 reverse transcriptase (RT) to mouse ornithine decarboxylase (ODC), a protein rapidly degraded by proteasome in an ubiquitine-independent fashion, to enhance the introduction of RT into the MHC class I pathway. We also designed a fusion of RT with two short signals from the C-terminus of ODC (ODCsig) representing a minimal proteasome-targeting moiety of ODC (PEST signal). Fusion to ODC or ODC signal domain led to a marked enhancement of RT degradation. Plasmids encoding RT-ODC and RT-ODCsig chimera were used to immunize BALB/c mice. The administration of the plasmids was not associated with autoimmune disease. Moreover, mice receiving RT-ODCsig gene mounted a mixed Th1/Th2 response characterized by the in vitro secretion of IFN-gamma, IL-2, TNF-alpha, IL-4, and IL-10 upon stimulation of splenocytes with RT protein or RT derived peptides. Serum titers of 10(2) to 10(3) were observed in more than 50% of animals in that group, whereas fewer animals mounted an anti-RT response in the RT-ODC gene immunized group. Chimeras of the type described here can, therefore, be used in vaccinations aiming to induce HIV-1 RT-specific immune response.
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PMID:HIV-1 reverse transcriptase artificially targeted for proteasomal degradation induces a mixed Th1/Th2-type immune response. 1846 38

Tristetraprolin (TTP) is a trans-acting factor that can regulate mRNA stability by binding to the cis-acting AU-rich element (ARE) in the 3'-untranslated region in mRNAs of certain transiently expressed genes. The best-studied target of TTP is tumor necrosis factor (TNF)-. By binding to ARE, TTP increases the degradation of TNF-alpha mRNA, thereby reducing the expression of TNF-alpha. We examined the effects of cAMP analogs and the cAMP-elevating agents forskolin and beta2-agonists on lipopolysaccharide (LPS)-induced TTP mRNA and protein expression by quantitative real-time reverse transcriptase-polymerase chain reaction and Western blotting in activated macrophages. All of these agents caused a slight increase in LPS-induced expression of TTP mRNA. However, TTP protein levels were significantly reduced when the cells were treated with the combination of LPS and cAMP-elevating agent compared with LPS alone. Proteasome inhibitors MG132 (N-[(phenylmethoxy)-carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide) and lactacystin increased TTP protein levels and abolished the effects of cAMP-enhancing compounds on TTP protein levels. The results suggest that mediators and drugs that enhance intracellular cAMP reduce TTP expression in macrophages exposed to inflammatory stimuli by increasing TTP degradation through the proteasome pathway.
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PMID:Compounds that increase or mimic cyclic adenosine monophosphate enhance tristetraprolin degradation in lipopolysaccharide-treated murine j774 macrophages. 1846 59

Skeletal muscle atrophy and fatty infiltration develop after tendon tearing. The extent of atrophy serves as one prognostic factor for the outcome of surgical repair of rotator cuff tendon tears. We asked whether mRNA of genes involved in regulation of degradative processes leading to muscle atrophy, ie, FOXOs, MSTN, calpains, cathepsins, and transcripts of the ubiquitin-proteasome pathway, are overexpressed in the supraspinatus muscle in patients with and without rotator cuff tears. We evaluated biopsy specimens collected during surgery of 53 consecutive patients with different sizes of rotator cuff tendon tears and six without tears. The levels of corresponding gene transcripts in total RNA extracts were assessed by semiquantitative reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Supraspinatus muscle atrophy was assessed by MRI. The area of muscle tissue (or atrophy), decreased (increased) with increasing tendon tear size. The transcripts of CAPN1, UBE2B, and UBE3A were upregulated more than twofold in massive rotator cuff tears as opposed to smaller tears or patients without tears. These atrophy gene products may be involved in cellular processes that impair functional recovery of affected muscles after surgical rotator cuff repair. However, the damaging effects of gene products in their respective proteolytic processes on muscle structures and proteins remains to be investigated.
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PMID:Expression of atrophy mRNA relates to tendon tear size in supraspinatus muscle. 1894 55

Advanced gallbladder cancer has an extremely poor prognosis because of metastasis. Identification of metastasis-related biomarkers is essential to improve patient survival. In the present study, metastasis-associated proteins were identified by comparative proteomic analysis and the metastasis-related function of the candidate protein, chloride intracellular channel 1 (CLIC1), was further elucidated. Two cell lines with high or low metastatic potential (termed GBC-SD18H and GBC-SD18L, respectively), originating from the same parental gallbladder carcinoma GBC-SD cell line, were identified by spontaneous metastasis in vivo and characterized by metastatic phenotypes analysis in vitro. Subsequently, a proteomic approach comprised of two-dimensional gel electrophoresis analysis and mass spectroscopy was used to identify and compare the protein expression patterns between GBC-SD18L and GBC-SD18H. Twenty-six proteins were identified and further verified by one-dimensional Western blotting and semiquantitative reverse transcriptase polymerase chain reaction analysis. It was determined that CLIC1, ezrin, vimentin, annexin A3, WD repeat domain 1, triosephosphate isomerase, C1-tetrahydrofolate synthase, Rho GDP-dissociation inhibitor 1, T-complex protein 1, heterogeneous nuclear ribonucleoprotein K, glutamate dehydrogenase 1, proteasome activator complex subunit 3 and Rab GDP-dissociation inhibitor beta were significantly up-regulated in the highly metastatic GBC-SD18H cell line compared to the poorly metastatic GBC-SD18L cell line. However, phosphoglycerate kinase 1 and programmed cell death protein 8 were significantly down-regulated in the highly metastatic GBC-SD18H cell line compared to GBC-SD18L. Considering that CLIC1 was profuse in highly metastatic GBC-SD18H but scarce in poorly metastatic GBC-SD18L, the association of CLIC1 with metastasis was further elucidated by the overexpression and RNA interference of CLIC1 in GBC-SD18L cells and GBC-SD18H cells, respectively. The results demonstrated that the overexpression of CLIC1 promoted cell motility and invasion of GBC-SD18L in vitro, while RNA interference of CLIC1 remarkably decreased cell motility and invasive potency of GBC-SD18H in vitro, indicating that CLIC1 might play an important role in metastasis of gallbladder carcinoma.
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PMID:Identification of metastasis-associated proteins involved in gallbladder carcinoma metastasis by proteomic analysis and functional exploration of chloride intracellular channel 1. 1929 76

There has been accumulating evidence for the involvement of retroviral integrase (IN) in the reverse transcription of viral RNA. We previously identified a host factor, survival motor neuron-interacting protein 1 (SIP1/Gemin2) that binds to human immunodeficiency virus type 1 (HIV-1) IN and supports HIV-1 infection apparently at reverse transcription step. Here, we demonstrated that HIV-1 IN together with SIP1 augments reverse transcriptase (RT) activity by enhancing the assembly of RT on viral RNA in vitro. Synthetic peptides corresponding to the binding motifs within IN that inhibited the IN-SIP1 interaction abrogated reverse transcription in vitro and in vivo. Furthermore, knockdown of SIP1 reduced intracellular stability and multimer formation of IN through proteasome-mediated degradation machinery. Taken together, SIP1 appears to stabilize functional multimer forms of IN, thereby promoting the assembly of IN and RT on viral RNA to allow efficient reverse transcription, which is a prerequisite for efficient HIV-1 infection.
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PMID:Augmentation of reverse transcription by integrase through an interaction with host factor, SIP1/Gemin2 Is critical for HIV-1 infection. 1991 60


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