Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.7.48 (transcriptase)
 9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The M1-type and M2-type isozymes of pyruvate kinase are produced from a single gene by mutually exclusive use of exons 9 and 10. Selection of exon 10 generates the M2 type, which occurs in most tissues, whereas the M1 type is expressed by use of exon 9 only in skeletal muscle, heart and brain. We investigated the mechanism by which exon 10, but not exon 9 is selected in M2-expressing cells by transfecting minigenes containing exon 9 and/or exon 10 in cells and by analyzing the transcripts using reverse-transcriptase polymerase chain reaction. Deletion of the most conserved region in intron 8 did not affect selection of exon 10 in dRLh-84 cells, which express only the M2 type. Exclusion of exon 10 from the minigene resulted in two major spliced products. One included correctly spliced exon 9 and the other skipped this exon. Similar splicing patterns were observed when these minigenes were transfected in hepatocytes which express the L type, but not M1 or M2 types. The 5' splice site but not the 3' splice site of exon 9 was found to be hardly recognized by the splicing machinery in dRLh-84 cells. Mutation of the 5' splice site sequence of exon 9 to that of exon 10 and vice versa did not change the splicing patterns. However, mutation of this site of exon 9 to a perfectly complementary sequence of U1 snRNA resulted in selection of exon 9 correctly spliced to exon 10. A 9-10 fusion exon (constructed by substitution of 68 bases of the 3' portion of exon 9 and 33 bases of the 5' portion of intron 9 for the corresponding regions of exon 10 and intron 10) was also correctly incorporated into a major product together with exon 10. Thus, we propose that exon 9 is not recognized in non M1-expressing cells due to the weak signal of its 5' splice site and that, although the 5' splicing signal of exon 10 also appears to be weak, this exon can be recognized in these cells because the 5' recognition signal may be relatively strengthened by cis-acting element(s) which may be present in the 3' portion of exon 9 and the 5' portion of intron 9 and/or the corresponding regions of exon 10 and intron 10.
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PMID:Alternative splicing of the pyruvate kinase M gene in a minigene system. 863 56

Stomach cancer is still a major cause of death in Asian people despite a complete cure after the resection of early cancers, mainly because peritoneal dissemination is difficult to treat. In the present study, we used two-dimensional differential gel electrophoresis (2-D DIGE) to identify specific proteins differentially expressed between a highly metastatic stomach cancer cell line MKN-45-P and its parental cell line MKN-45. We detected 27 protein spots in at least 2 of 3 experiments which showed statistically significant differences in abundance. All 27 protein spots were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) and database-searching software. A proteomic analysis revealed 13 different proteins with some isoforms sharing different biochemical characteristics, and that 8 proteins were up-regulated, and 5 were down-regulated. The 13 proteins were mainly involved in protein synthesis (transfer RNA synthetase), metabolism (flavoprotein subunit, pyruvate kinase, adenylate kinase), receptor and signal transduction (annexins I and A2), the cytoskeleton (keratin 5, cytokeratin 8) and cell cycling (ts11). These results suggested that a proteomic approach including 2-D DIGE would be an efficient way to identify the proteins responsible for specific biological functions. Moreover, these observations might be novel findings leading to the prediction of postoperative peritoneal recurrence.
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PMID:Proteomic analysis of a highly metastatic gastric cancer cell line using two-dimensional differential gel electrophoresis. 1696 83

Tetramethylpyrazine is the active ingredient of a Chinese herbal medicine. In this study, tetramethylpyrazine was tested for its activities in irradiated bone marrow stromal QXMSC1 cells. The proliferation of QXMSC1 cells was measured by MTS assay kit and flow cytometry. To identify proteins involved in the processes of cellular and molecular response of tetramethylpyrazine to irradiation damage, we comparatively analyzed the proteome of nonirradiated, irradiated and tetramethylpyrazine treated QXMSC1 cells. Reverse transcriptase polymerase chain reaction (RT-PCR) were used to validate the differentially expressed proteins. 20 Gy 60Co gamma irradition inhibited QMSC1 cells growth and tetramethylpyrazine could reverse of this action due to stimulating QXMSC1 cells from G1 to S progression. Proteomic analytical results showed that 18 spots were changed in irradiated QXMSC1 cells, and 15 spots matched with known proteins after database searching. The expression level of proteins such as translationally controlled tumor protein (TCTP), and galectin-3, were increased in irradiated QXMSC1 cells, while calmodulin, pyruvate kinase were decreased. Tetramethylpyrazine could prevent this change or reverse to some degree. The function of these proteins involves in hematopoiesis, cell cycle and signal transduction. The changes of these proteins were confirmed by RT-PCR at mRNA levels. This study suggested that stimulating proliferation via tetramethylpyrazine played an important role in the cure effect on irradiated QXMSC1 cells and was helpful to deeply understand the mechanism of tetramethylpyrazine at the molecular level.
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PMID:Proteomic analysis of the effects of tetramethylpyrazine on irradiated QXMSC1 cells. 1726 90

Hepatitis C virus (HCV) replication and pathogenesis involve both virus-encoded proteins and cellular factors. In our study, we showed that NS5B, the HCV RNA-dependent RNA polymerase, interacted with M2 type pyruvate kinase (M2PK) but not L type pyruvate kinase. We confirmed the interaction by GST pull down, coimmunoprecipitation and confocal immunofluorescence analysis in cells with transient expression of NS5B and M2PK as well as in a HCV replicon-bearing cell line. Furthermore shRNA which specifically down-regulated M2PK expression could inhibit the replication of HCV in HCV replicon 9B cells.
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PMID:Isoform-specific interaction of pyruvate kinase with hepatitis C virus NS5B. 1851 40

Influenza virus RNA-dependent RNA polymerase (RdRp) is a heterotrimer of three viral proteins, PB1, PB2, and PA and is involved in both transcription and replication of the negative strand of the viral RNA (vRNA) genome. RdRp is multifunctional, possessing RNA polymerase, cap binding, and endonuclease activities. The enzyme synthesizes three different RNAs, complementary RNA (cRNA) and messenger RNA (mRNA) from vRNA, and vRNA from cRNA. To synthesize these three RNAs, RdRp requires conversion of its function by host factor. Here, we performed yeast two-hybrid screening to identify the relevant host factor, revealing that pyruvate kinase M2 (PKM2) interacted with the PA subunit of influenza virus RdRp. PKM2 is one of two enzymes (PKM1 and PKM2) produced by alternative splicing of the pyruvate kinase M (PKM) pre-mRNA. We determined the interacting regions in both PKM2 and PA, the expression level of PKM by western blotting at different time points after viral infection, and the effects of transfection of siRNA targeting PKM on influenza virus replication. The results demonstrated that the C-terminal region of PKM2 interacted with the C-terminus of the PA subunit, that the expression level of PKM2 increased with influenza virus infection time, and that this enzyme is essential for influenza virus multiplication. Moreover, isoelectric focusing of uninfected and influenza virus infected cell extracts, followed by gradient gel electrophoresis to separate the PKM1 and PKM2 isoforms and western blotting indicated that PKM2 became more acidic after influenza infection. The decreased pH of PKM2 may have been due to phosphorylation, and phosphorylated PKM2 is active as a pyruvate kinase and protein kinase; therefore, it is possible that PKM2 may transfer a phosphate group to PA and consequently transform the function of RdRp from transcriptase to replicase.
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PMID:Influenza Virus Infection Induces Host Pyruvate Kinase M Which Interacts with Viral RNA-Dependent RNA Polymerase. 2823 20