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Proteins involved in sleep homeostasis: biophysical characterization of INC and its partners

DOI: 10.1016/j.biochi.2016.09.013 DOI Help

Authors: Luciano Pirone (Institute of Biostructures and Bioimaging, C.N.R.) , Giovanni Smaldone (IRCCS SDN) , Carla Esposito (Institute of Biostructures and Bioimaging, C.N.R.) , Nicole Balasco (Institute of Biostructures and Bioimaging, C.N.R.) , Maxim V. Petoukhov (European Molecular Biology Laboratory) , Alessandro Spilotros (European Molecular Biology Laboratory) , Dmitri I. Svergun (European Molecular Biology Laboratory) , Sonia Di Gaetano (Institute of Biostructures and Bioimaging, C.N.R.) , Luigi Vitagliano (Institute of Biostructures and Bioimaging, C.N.R.) , Emilia Maria Pedone (Institute of Biostructures and Bioimaging, C.N.R.)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochimie

State: Published (Approved)
Published: September 2016

Abstract: The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.

Journal Keywords: Protein-protein interactions; Protein stability; Binding; Homologs; Solution scattering

Subject Areas: Biology and Bio-materials

Instruments: B21-High Throughput SAXS

Added On: 01/10/2016 03:21

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