ABSTRACT
Placental OCTN2 is a high-affinity carnitine transporter that can interact with a number of therapeutic agents. The process of syncytialization is associated with the expression of a variety of genes. However, the association between syncytialization and OCTN2 expression is not yet clear. Given that forskolin induces BeWo cells to undergo biochemical and morphological differentiation, the purpose of the present study was to investigate whether the function and expression of OCTN2 are influenced by forskolin treatment during syncytialization. The forskolin-induced differentiation of BeWo cells was validated by secretion of beta-human chorionic gonadotropin (beta-hCG) and syncytin expression. Cellular localization of OCTN2 was analyzed by confocal microscopy. Expression of OCTN2 and the modular proteins PDZK1, PDZK2, NHERF1 and NHERF2 was analyzed by Western blotting and carnitine uptake by BeWo cells was estimated and the kinetic properties of uptake measured. The results showed that forskolin treatment increased beta-hCG secretion and syncytin expression, suggesting induction of syncytialization. Confocal images of BeWo cells showed the localization of OCTN2 in the brush-border membrane. OCTN2 protein expression was upregulated in isolated brush-border membranes by long-term forskolin treatment, but the V(m) for carnitine uptake was unchanged, although the K(m) increased. PDZK1, NHERF1 and NHERF2 protein expression in the brush-border membrane was downregulated by forskolin treatment, whereas PDZK2 levels remained unchanged. In conclusion, protein expression and function of OCTN2 in BeWo cells can be regulated by forskolin treatment. While the presence of forskolin results in an increase in OCTN2 protein expression, the increase in uptake capacity may be compensated by the decreased expression of PDZK1, NHERF1 or NHERF2.
Subject(s)
Colforsin/pharmacology , Giant Cells/drug effects , Organic Cation Transport Proteins/metabolism , Trophoblasts/drug effects , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Transformation, Neoplastic , Choriocarcinoma/pathology , Chorionic Gonadotropin, beta Subunit, Human/metabolism , Gene Expression Regulation/drug effects , Gene Products, env/genetics , Gene Products, env/metabolism , Giant Cells/metabolism , Humans , Membrane Proteins , Microvilli/drug effects , Microvilli/metabolism , Organic Cation Transport Proteins/genetics , Phosphoproteins/genetics , Phosphoproteins/metabolism , Pregnancy Proteins/genetics , Pregnancy Proteins/metabolism , Sodium-Hydrogen Exchangers/genetics , Sodium-Hydrogen Exchangers/metabolism , Solute Carrier Family 22 Member 5 , Trophoblasts/metabolism , Trophoblasts/pathologyABSTRACT
tRNA-guanine transglycosylase (TGT) is a key enzyme involved in the posttranscriptional modification of tRNA across the three kingdoms of life. In eukaryotes and eubacteria, TGT is involved in the introduction of queuine into the anticodon of the cognate tRNAs. In archaebacteria, TGT is responsible for the introduction of archaeosine into the D-loop of the appropriate tRNAs. The tRNA recognition patterns for the eubacterial (Escherichia coli) TGT have been studied. These studies are all consistent with a restricted recognition motif involving a U-G-U sequence in a seven-base loop at the end of a helix. While attempting to investigate the potential of negative recognition elements in noncognate tRNAs via the use of chimeric tRNAs, we have discovered a second recognition site for the E. coli TGT in the TpsiC arm of in vitro-transcribed yeast tRNA(Phe). Kinetic analyses of synthetic mutant oligoribonucleotides corresponding to the TpsiC arm of the yeast tRNA(Phe) indicate that the specific site of TGT action is G53 (within a U-G-U sequence at the transition of the TpsiC stem into the loop). Posttranscriptional base modifications in tRNA(Phe) block recognition by TGT, most likely due to a stabilization of the tRNA structure such that G53 is inaccessible to TGT. These results demonstrate that TGT can recognize the U-G-U sequence within a structural context that is different than the canonical U-G-U in the anticodon loop of tRNA(Asp). Although it is unclear if this second recognition site is physiologically relevant, this does suggest that other RNA species could serve as substrates for TGT in vivo.
Subject(s)
Escherichia coli/enzymology , Pentosyltransferases/metabolism , RNA, Transfer, Phe/metabolism , Base Sequence , Binding Sites/genetics , Enzyme Stability , Escherichia coli/genetics , Kinetics , Macromolecular Substances , Molecular Sequence Data , Nucleic Acid Conformation , RNA/chemistry , RNA/genetics , RNA/metabolism , RNA Processing, Post-Transcriptional , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , RNA, Bacterial/metabolism , RNA, Fungal/chemistry , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Transfer, Phe/chemistry , RNA, Transfer, Phe/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Substrate SpecificityABSTRACT
A key enzyme involved in the incorporation of the modified base queuine into tRNA (position 34) is tRNA-guanine transglycosylase (TGT). Studies of the recognition of truncated tRNAs by the Escherichia coli TGT have established a minimal recognition motif involving a minihelix with a 7 base loop containing a U-G-U sequence (where G is replaced with queuine) [Curnow, A.W. and Garcia, G.A. (1995) J. Biol. Chem. 270, 17264-17267; Nakanishi, S. et al. (1994) J. Biol. Chem. 269, 32221-32225]. Still, a clearer understanding of the recognition of full-length 'queuine-cognate' tRNAs by TGT remains lacking. In this paper, we report the in vitro transcription and enzymological characterization (Km, and kcat) of all four 'queuine-cognate' tRNAs from E. coli and from Saccharomyces cerevisiae with the TGT from E. coli. No primary or secondary structures emerge as important recognition elements from this study. The modest differences in substrate specificity (relative kcat/Km values vary from 0.5 to 8.4) seen among these 'queuine-cognate' tRNAs most likely result from the accumulated effects of many subtle factors. Interestingly, the yeast tRNAs are essentially equivalent to the E. coli tRNAs as substrates for TGT, indicating that there is nothing intrinsic to the yeast tRNAs that accounts for the absence of queuine in yeast.
Subject(s)
Escherichia coli/enzymology , Guanine/analogs & derivatives , Pentosyltransferases/metabolism , RNA, Transfer/metabolism , RNA-Binding Proteins/metabolism , Base Sequence , Guanine/metabolism , Kinetics , Nucleic Acid Conformation , Plasmids , Polymerase Chain Reaction , RNA, Transfer/chemistry , RNA, Transfer/genetics , Substrate Specificity , Templates, Genetic , Transcription, GeneticABSTRACT
tRNA-guanine transglycosylase (TGT) is the enzyme responsible for the post-transcriptional modification of specific tRNAs (those for Asn, Asp, His, and Tyr) with the hypermodified base, queuine. In Escherichia coli this enzyme catalyzes the exchange of guanine-34 in the anticodon with preQ1, which is subsequently further modified to queuine. There is evidence that such hypermodified tRNA molecules may play a role in the control of cell proliferation and differentiation. In order to perform detailed, in vitro mechanistic studies and to probe the tRNA-enzyme interaction, we have generated unmodified E. coli tRNA(Tyr) and truncated analogues using an in vitro RNA synthesis system suggested by Milligan and Uhlenbeck [Milligan, J. F., & Uhlenbeck, O. C. (1989) Methods Enzymol. 180, 51-62]. From this system we have generated three tRNA analogues totally devoid of any post-transcriptional modifications. In order to compare the unmodified tRNA with the true physiological substrate for TGT, that is, tRNA that contains all modified bases except queuine, we have isolated E. coli tRNA(Tyr) from an overexpressing clone in a TGT-deficient strain of E. coli. We report here that unmodified, full-length tRNA(Tyr) serves as a substrate for TGT with kinetic parameters that are, within experimental error, the same as those for in vivo isolated tRNA(Tyr). This indicates that other post-transcriptional modifications have negligible effects upon TGT recognition of tRNA. A 17-base oligoribonucleotide, corresponding to the anticodon loop and stem, is also a substrate for TGT with only a 20-fold loss in Vmax/KM, versus the full-length tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)
