RESUMO
Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.
Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , Linhagem Celular , Proliferação de Células , Galinhas , DNA Helicases/deficiência , DNA Helicases/genética , Primers do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases , Humanos , Técnicas In Vitro , Camundongos , RecQ Helicases , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Troca de Cromátide Irmã , Técnicas do Sistema de Duplo-Híbrido , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de WernerRESUMO
The current therapeutic angiogenesis strategy to treat ischemic disease by using angiogenic growth factors has been limited to use of a single gene. However, as vasodilator substances such as prostacyclin are widely used for the treatment of peripheral arterial disease, it might be useful to combine angiogenesis with vasodilation of new vessels. In a mouse hind limb ischemia model, cotransfection of the hepatocyte growth factor (HGF) gene with the prostacyclin synthase gene demonstrated a further increase in blood flow and capillary density compared with a single gene. Even in the rabbit ischemia model, cotransfection of HGF plasmid with the prostacyclin synthase gene demonstrated a further increase in angiogenic activity compared with HGF alone. Because peripheral neuropathy due to diabetes is common for significant morbidity, we examined the hypothesis that experimental diabetic neuropathy can be reversed by HGF and prostacyclin synthase genes. Severe peripheral neuropathy, characterized by significant slowing of nerve conduction velocity compared with nondiabetic control animals, was ameliorated. Overall, cotransfection of the prostacyclin synthase and HGF genes is more effective than single-gene transfection to stimulate angiogenesis, and it significantly improved neuropathy. These data provide important information relating to the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.