title: Molecular characterization of HIV-1 Nef and ACOT8 interaction: insights from in silico structural predictions and in vitro functional assays.
abstract: HIV-1 Nef interacts with several cellular proteins, among which the human peroxisomal thioesterase 8 (ACOT8). This interaction may be involved in the endocytosis regulation of membrane proteins and might modulate lipid composition in membrane rafts. Nef regions involved in the interaction have been experimentally characterized, whereas structural details of the ACOT8 protein are unknown. The lack of structural information hampers the comprehension of the functional consequences of the complex formation during HIV-1 infection. We modelled, through in silico predictions, the ACOT8 structure and we observed a high charge complementarity between Nef and ACOT8 surfaces, which allowed the identification of the ACOT8 putative contact points involved in the interaction. The predictions were validated by in vitro assays through the development of ACOT8 deletion mutants. Coimmunoprecipitation and immunofluorescence analyses showed that ACOT8 Arg(45)-Phe(55) and Arg(86)-Pro(93) regions are involved in Nef association. In addition, K91S mutation abrogated the interaction with Nef, indicating that Lys(91) plays a key role in the interaction. Finally, when associated with ACOT8, Nef may be preserved from degradation. These findings improve the comprehension of the association between HIV-1 Nef and ACOT8, helping elucidating the biological effect of their interaction.
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy.
authors.forename: Michela
authors.initials: M
authors.lastname: Serena
authors.affiliation: Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Giorgetti
authors.affiliation: Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy.
authors.forename: Mirko
authors.initials: M
authors.lastname: Busato
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy.|Department of Biotechnology, University of Verona, Strada le Grazie 15, 37134 Verona, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Gasparini
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy.
authors.forename: Erica
authors.initials: E
authors.lastname: Diani
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy.
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Romanelli
authors.affiliation: Department of Neurosciences, Biomedicine and Movement Sciences, University of Verona, Strada le Grazie 8, 37134 Verona, Italy.
authors.forename: Donato
authors.initials: D
authors.lastname: Zipeto
mesh_terms: D002460:Cell Line; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D008239:Lysine / Q000235:genetics; D008958:Models, Molecular; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D017384:Sequence Deletion / Q000235:genetics; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; C106353:ACOT8 protein, human; D010170:Palmitoyl-CoA Hydrolase; D008239:Lysine