RESUMO
The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Catálise , Ciclo Celular , Quinases Ciclina-Dependentes/metabolismo , Células HeLa , Humanos , Fosforilação , Poliubiquitina/metabolismo , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Supressoras de Tumor , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/químicaRESUMO
Cardiopulmonary bypass (CPB) circuits are frequently necessary in the repair of congenital heart defects in infants and children. Although advances in technology and operative technique have decreased the mortality associated with cardiac procedures requiring CPB, post-operative neuro-cognitive outcome and the role of the CPB circuit in post-operative morbidity remains a significant concern. There are several factors that have been suggested to play a significant role in general post-operative outcome, including intraoperative inflammatory responses caused by the interaction of blood with circuit component surfaces, selection of appropriate perfusion mode to optimize organ function during CPB, and the introduction of gaseous microemboli into the patient's systemic circulation through circuit manipulations and modifications. These factors are the subject of continuing research at the Penn State Hershey Children's Hospital Pediatric Cardiac Research Laboratories, and this review will focus on the results of studies aimed at identifying circuit elements that affect the delivery of gaseous microemboli to the patient during CPB procedures, the role of anti-factor D monoclonal antibody in reducing systemic inflammation during CPB, and the results of preliminary plasma proteomics studies conducted on infants undergoing CPB.