title: Proteomic analysis of the cullin 4B interactome using proximity-dependent biotinylation in living cells.
abstract: Cullin 4B (CUL4B) mutations have been implicated in mental retardation and dopamine-related behaviors due to disruptions in their interaction with cullin-RING E3 ligases (CRLs). Thus, further identification of CUL4B substrates can increase the knowledge of protein homeostasis and illuminate the role of CUL4B in neuropsychiatric disease. However, the transient nature of the coupling between CUL4B and its substrates is difficult to detect in vivo using current approaches, thus hampers efforts to investigate functions of CRLs within unperturbed living systems. In this study, we sought to discover CUL4B interactants with or without dopamine stimulation. BirA (118G) proximity-dependent biotin labeling combined with LC-MS was employed to biotinylate and identify transient and weak interactants of CUL4B. After purification with streptavidin beads and identified by LC-MS, a total of 150 biotinylated proteins were identified at baseline condition, 53 of which are well-known CUL4B interactants. After dopamine stimulation, 29 proteins disappeared and were replaced by 21 different protein interactants. The altered CUL4B interactants suggest that CUL4B regulates protein turnover and homeostasis in response to dopamine stimulation. Our results demonstrate the potential of this approach to identify novel CUL4B-related molecules in respond to cellular stimuli, which may be applied to other types of signaling pathways.
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
authors.forename: Hailong
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.|Department of Psychiatry, University of Toronto, Toronto, ON, Canada.
authors.forename: Shupeng
authors.initials: S
authors.lastname: Li
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
authors.forename: Pingting
authors.initials: P
authors.lastname: Liu
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.
authors.forename: Frankie H F
authors.initials: FHF
authors.lastname: Lee
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.|Department of Psychiatry, University of Toronto, Toronto, ON, Canada.
authors.forename: Albert H C
authors.initials: AHC
authors.lastname: Wong
authors.affiliation: Campbell Research Institute, Centre for Addiction and Mental Health, Toronto, ON, Canada.|Department of Psychiatry, University of Toronto, Toronto, ON, Canada.
authors.forename: Fang
authors.initials: F
authors.lastname: Liu
mesh_terms: D019921:Biotinylation / Q000187:drug effects; D019731:Carbon-Nitrogen Ligases / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism*; D004298:Dopamine / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods; D060066:Protein Interaction Maps; D040901:Proteomics / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results
chemical_list: C517832:CUL4B protein, human; D044842:Cullin Proteins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D004298:Dopamine
keywords: Biotinylation; CRLs proximal labeling; Cellular stimuli; Interactants; Protein dynamics