Proteins involved in sleep homeostasis: Biophysical characterization of INC and its partners.
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.
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy.
Luciano
L
Pirone
IRCCS SDN, Via Emanuele Gianturco 113, 80143 Napoli, Italy.
Giovanni
G
Smaldone
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy.
Carla
C
Esposito
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy.
Nicole
N
Balasco
European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.
Maxim V
MV
Petoukhov
European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.
Alessandro
A
Spilotros
European Molecular Biology Laboratory, Hamburg Outstation, c/o DESY, Notkestrasse 85, 22607 Hamburg, Germany.
Dmitri I
DI
Svergun
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address: digaetan@unina.it.
Sonia
S
Di Gaetano
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address: luigi.vitagliano@unina.it.
Luigi
L
Vitagliano
Institute of Biostructures and Bioimaging, C.N.R., Via Mezzocannone 16, 80134 Napoli, Italy. Electronic address: empedone@unina.it.
Emilia Maria
EM
Pedone
D000595:Amino Acid Sequence; D000818:Animals; D055592:Biophysical Phenomena; D002942:Circular Dichroism; D044842:Cullin Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D000077614:Golgi Matrix Proteins; D006706:Homeostasis; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D053838:Scattering, Small Angle; D017386:Sequence Homology, Amino Acid; D012890:Sleep / Q000502:physiology*; D013696:Temperature; D014961:X-Ray Diffraction
C503647:Cul3 protein, Drosophila; D044842:Cullin Proteins; D029721:Drosophila Proteins; C506558:GRASP65 protein, Drosophila; D000077614:Golgi Matrix Proteins; C569969:Inc protein, Drosophila; D008565:Membrane Proteins
Binding; Homologs; Protein stability; Protein-protein interactions; Solution scattering