title: Functional characterisation of the WW minimal domain for delivering therapeutic proteins by adenovirus dodecahedron.
abstract: Protein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron) has recently been shown to deliver proteins fused to the tandem WW(2-3-4) structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW(3) and its mutant form WW(3)_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application.
authors.affiliation: HumProTher Laboratory, TheREx, TIMC-IMAG Laboratory, CNRS UMR5525, University Joseph Fourier, La Tronche, France.
authors.forename: Ana
authors.initials: A
authors.lastname: Villegas-Méndez
authors.forename: Pascal
authors.initials: P
authors.lastname: Fender
authors.forename: Marina I
authors.initials: MI
authors.lastname: Garin
authors.forename: Romy
authors.initials: R
authors.lastname: Rothe
authors.forename: Lavinia
authors.initials: L
authors.lastname: Liguori
authors.forename: Bruno
authors.initials: B
authors.lastname: Marques
authors.forename: Jean-Luc
authors.initials: JL
authors.lastname: Lenormand
mesh_terms: D000256:Adenoviridae / Q000235:genetics*; D017209:Apoptosis / Q000235:genetics / Q000502:physiology; D015153:Blotting, Western; D045744:Cell Line, Tumor; D024202:Electrophoretic Mobility Shift Assay; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D045325:HCT116 Cells; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53