Inhibition of E3 ubiquitin ligase Wwp1 with selective DNA aptamers
Wesley Tucker
Hong Kong University,Li Ka Shing Faculty of Medicine, 21 Sassoon Road, Pokfulam, Hong Kong
Abstract:
WWP1 is a Nedd4 family ubiquitin ligase known to complex with Schnurri3 and polyubiquitinate Runx2, a key transcriptional factor associated with osteoblast differentiation. Runx2 is therefore directed to the proteosome, making WWP1 and Shnurri3 negative regulators of Runx2 and osteoblast differentiation. Hence, the disruption of this complex could impede the polyubiquitination of Runx2, causing an increase in osteoblast growth and thus stronger bone in patients suffering from osteoporosis. To this end, three truncations of WWP1 have been cloned into expression vectors, expressed in E. coli, and purified using poly-Histidine tag chromatography. These truncations are currently being used to select highly specific DNA aptamers with the established SELEX method which will tightly bind particular domains of WWP1. Pools of selected aptamers will be characterized for homogeneity with DNA sequencing, secondary structure with Circular Dichromism, and binding strength with Isothermal Titration Calorimetry. Selected aptamers will then be modified for stability in the cell for In Vitro cell culture models, in view of the delivery by transfection to tissues in an In Vivo model. This work should lay a foundation for the unmet need of an improved osteoporosis therapy in the future.
