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Defining the α-synuclein interactome In situ
Xiuqi Cao
Undergraduate Student at Yale University B.S. in Molecular, Cellular & Developmental Biology, 2017
Mentor: Karina Vargas
PI: Sreeganga S. Chandra
Clayton DF, George JM.
J Neurosci Res. 1999
Background
• Localized in presynaptic terminal and implicated in many parts of synaptic vesicle cycle
• The function of α-synuclein
is poorly understood because of its conformational plasticity
• Previous α-synuclein overexpression experiments may have lead to pathological rather than physiological response
Chandra S. (2009) Encyclopedia of Neuroscience
Unfolded α-Synuclein Oligomeric α-Synuclein
Helical Conformation α-Synuclein Fibrils
Using abg-Synuclein Triple-Knockout (TKO) Mice to Pinpoint Synuclein Function
Greten-Harrison et al. (2010) PNAS 107:19573-78
TKO Leads to Slowed Endocytosis that can be Rescued via Synuclein Expression
Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376
Synucleins Implicated in Clathrin Mediated Endocytosis
Karina J. Vargas et al. J. Neurosci. 2014;34:9364-9376
Proximal Protein Labeling
Purpose: Define the normal function of α-synuclein through determining its protein interactome
Proximity-Dependent Biotin Identification via BirA* Fusion
Kyle J. Roux et al. J Cell Biol 2012;196:801-810
Synuclein-BirA* has Limited Biotinylation in Differentiated SH-SY5Y Cells
α-synuclein
biotin
55 kDa
55 kDa
55 kDa myc
Wang et al, Curr. Biol 2014
Burre et al, J Neurosci 2015
Proximity-Dependent Biotin Identification via APEX2 Fusion
Stephanie S. Lam et al. Nature Methods 2015;12:51-54
APEX2 biotinylates stronger and faster in 1 minute than BirA* in 24 hours
Ongoing Studies
We will be reconstituting the APEX2-synuclein fusion protein with isolated PTK2 membrane and whole brain cytosol to attempt biotinylation of endocytic proteins.
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