| Tyrosinase (EC1.14.18.1) is a copper-containing oxidase, widely found inmicroorganisms, plants, animals and humans.It is the rate-limiting enzyme in melaninformation.Tyrosinase mutation causes the hair, skin, eyes hypopigmentation.Tyrosinaseis not the only regulator in melanin formation and melanocytes apoptosis,it needs tobind with other protein factor.The purpose of this project is to find new tyrosinasebinding protein factors and elucidate its mechanism.It is important to study theregulatory mechanism of melanin formation.The project is based on ten candidate genesthat were found through yeast two-hybrid previously.Analysis by simulating moleculardocking.Finally selecting Tyrp1and GIPC1proteins as the research subjects.The use ofco-immunoprecipitation and GST pull-down method to determine the interactionbetween tyrosinase and Tyrp1,so as tyrosinase and GIPC1,clarify its role in theregulation of tyrosinase.The study found that not only the new regulatory factors inmelanin formation,but also helps to provide new clues to human-related skin diseases.1.Create a three-dimensional structure model of human tyrosinase,predicte108proteins that are directly and indirectly interacted with tyrosinase in melaninformation.The study found G protein-associated proteins,Potassium channel-associatedproteins,Vesicle/sorting-associated proteins,are with a stronger interaction withtyrosinase.2.Use PEIMAP algorithm selected five kinds of high scoring protein GIPC1,GIPC2, GIPC3, DCT, Tyrp1docking with tyrosinase.Found the five proteins’ dockingregion is the active center of tyrosinase.3.Construct tyrosinase eukaryotic expresstion vector with SRT label.Gained C89Rand R299H mutant that have a big influence on tyrosinase.4.Gained pCMV-Flag-Pxy(Pxy means Unknown protein factor) andpCMV6-SRT-Tyr vectors Cotransfected HEK293cell.Purified His-GIPC1,His-Tyrp1and GST-Tyr proteins from E. coli cells. 5.Verify the interaction between Tyrosinase and the targeted protein GIPC1,Tyrp1by GST pull-down and co-immunoprecipitation.GST pull-down show GIPC1and Tyrp1are binding with Tyrosinase in vitro.co-immunoprecipitation show GIPC1is bindingwith Tyrosinase in vivo. |
