RESUMO
Poly(ADP-ribose) polymerases (PARPs) comprise a growing family of enzymes known to be involved in genotoxic signaling and metabolic regulation. One of the latest family members, tankyrase 1, was shown to be involved in maintenance of telomere integrity. Here we expressed full-length tankyrase 1 and a fragment, termed T-PARP, spanning the poly(ADP-ribose) polymerase domain and characterized the enzymatic properties of the two proteins. Both, tankyrase 1 and T-PARP catalyze an auto poly(ADP-ribosyl)ation reaction with comparable catalytic activity. In contrast, (ADP-ribosyl)ation of TRF1, a previously described substrate, is strongly performed only by the full-length enzyme but not by T-PARP. Characterization of the poly(ADP-ribose) products reveals that tankyrase 1 synthesizes polymers with an average chain length of 20 units and no detectable branching of the polymers. Finally, we show that the catalytic efficiency of tankyrase 1, as expressed by the k(cat)/K(m) value, is approximately 150-fold lower compared to the basal activity of the poly(ADP-ribose) polymerase, PARP 1.
Assuntos
Fragmentos de Peptídeos/metabolismo , Tanquirases/metabolismo , Telômero/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , NAD/metabolismo , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tanquirases/química , Tanquirases/genética , Proteína 1 de Ligação a Repetições Teloméricas/genética , Proteína 1 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Telomerase, a ribonucleoprotein acting as a reverse transcriptase, has been identified as a target for cancer drug discovery. The synthetic, non-nucleosidic compound, BIBR1532, is a potent and selective telomerase inhibitor capable of inducing senescence in human cancer cells (). In the present study, the mode of drug action was characterized. BIBR1532 inhibits the native and recombinant human telomerase, comprising the human telomerase reverse transcriptase and human telomerase RNA components, with similar potency primarily by interfering with the processivity of the enzyme. Enzyme-kinetic experiments show that BIBR1532 is a mixed-type non-competitive inhibitor and suggest a drug binding site distinct from the sites for deoxyribonucleotides and the DNA primer, respectively. Thus, BIBR1532 defines a novel class of telomerase inhibitor with mechanistic similarities to non-nucleosidic inhibitors of HIV1 reverse transcriptase.