RESUMEN
Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.
Asunto(s)
Fosfatos de Dinucleósidos/química , Poli A/química , Polinucleotido Adenililtransferasa/química , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/efectos de los fármacos , Proteínas Bacterianas/metabolismo , Catálisis/efectos de los fármacos , Sistema Libre de Células , Fosfatos de Dinucleósidos/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Escherichia coli , Cinética , Poli A/biosíntesis , Polinucleotido Adenililtransferasa/efectos de los fármacos , Polinucleotido Adenililtransferasa/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The enzyme poly(A) polymerase (PAP; EC 2.7.7.19) catalyzes the polyadenylation of mRNAs. It's activity levels and isoforms vary within the cell cycle (31) and apoptosis (34). The direct effect of most anticancer drugs is cell damage (DNA and RNA synthesis inhibition, DNA breaks and/or cell cycle aberrations), which then triggers signaling pathways that activate apoptosis and eventually lead to regulated cell death. The experiments described here concern the chemotherapeutic agents, interferon (IFN) and 5-fluorouracil (5-FU), and their action on U937 cells, alone or in various combinations, resulting in the commitment of cell apoptosis, as observed by the appearance of DNA fragmentation. Furthermore, examination of U937 cell apoptotic trend in parallel with PAP activity measurements and isoforms detection by immunoblotting, revealed both partial enzyme inactivation and dephosphorylation, in particular after the combined drug action of 5-FU and IFN on U937 cells. Our work on chemotherapeutic drug action at the level of mRNA polyadenylation may contribute to new insights into the mechanism of cell apoptosis, as well as provide information on mRNA poly(A) tail formation, removal and function.
Asunto(s)
Apoptosis/fisiología , Fluorouracilo/farmacología , Interferón-alfa/farmacología , Polinucleotido Adenililtransferasa/metabolismo , Apoptosis/efectos de los fármacos , Citoplasma/enzimología , Fragmentación del ADN/efectos de los fármacos , Humanos , Isoenzimas/efectos de los fármacos , Isoenzimas/aislamiento & purificación , Isoenzimas/metabolismo , Cinética , Polinucleotido Adenililtransferasa/efectos de los fármacos , Polinucleotido Adenililtransferasa/aislamiento & purificación , Células U937RESUMEN
VP39 is a single-domain, bifunctional viral protein, which acts at both ends of nascent mRNA. At the 5' end, it acts as a cap-specific 2'-O-methyltransferase. At the 3' end, it acts as a poly(A) polymerase processivity factor, requiring its direct association with poly(A) polymerase. Although crystallographic and biochemical data show the catalytic center and associated binding sites for VP39's methyltransferase function to be juxtaposed around a superficial cleft on the protein surface, surface regions required for VP39's mRNA 3' end modifying functions are not known. Here, we identify a surface region that interfaces directly with poly(A) polymerase, taking three independent approaches: (i) development of a direct in vitro dimerization assay, which is applied to numerous VP39 point mutants; (ii) identification of sites within VP39 that become protected from protease cleavage upon dimerization and further mutagenesis based upon these data; (iii) site-specific photo-cross-linking of VP39 to VP55. We find that the dimerization interface lies on a surface region remote from the methyltransferase cleft and contains a 3-5-residue "hot-spot," which is very sensitive to amino acid substitutions. Various other sites within VP39 consistently became hypersensitive to protease cleavage upon interaction with VP55, indicating the occurrence of extensive conformational changes.