Profiling of Parkin-binding partners using tandem affinity purification.
Parkinson's disease (PD) is a progressive neurodegenerative disorder affecting approximately 1-2% of the general population over age 60. It is characterized by a rather selective loss of dopaminergic neurons in the substantia nigra and the presence of α-synuclein-enriched Lewy body inclusions. Mutations in the Parkin gene (PARK2) are the major cause of autosomal recessive early-onset parkinsonism. The Parkin protein is an E3 ubiquitin ligase with various cellular functions, including the induction of mitophagy upon mitochondrial depolarizaton, but the full repertoire of Parkin-binding proteins remains poorly defined. Here we employed tandem affinity purification interaction screens with subsequent mass spectrometry to profile binding partners of Parkin. Using this approach for two different cell types (HEK293T and SH-SY5Y neuronal cells), we identified a total of 203 candidate Parkin-binding proteins. For the candidate proteins and the proteins known to cause heritable forms of parkinsonism, protein-protein interaction data were derived from public databases, and the associated biological processes and pathways were analyzed and compared. Functional similarity between the candidates and the proteins involved in monogenic parkinsonism was investigated, and additional confirmatory evidence was obtained using published genetic interaction data from Drosophila melanogaster. Based on the results of the different analyses, a prioritization score was assigned to each candidate Parkin-binding protein. Two of the top ranking candidates were tested by co-immunoprecipitation, and interaction to Parkin was confirmed for one of them. New candidates for involvement in cell death processes, protein folding, the fission/fusion machinery, and the mitophagy pathway were identified, which provide a resource for further elucidating Parkin function.
Center for Biomedicine, European Academy Bozen/Bolzano (EURAC), Bolzano, Italy, Affiliated Institute of the University of Lübeck, Lübeck, Germany.
Alessandra
A
Zanon
Aleksandar
A
Rakovic
Hagen
H
Blankenburg
Nadezhda T
NT
Doncheva
Christine
C
Schwienbacher
Alice
A
Serafin
Adrian
A
Alexa
Christian X
CX
Weichenberger
Mario
M
Albrecht
Christine
C
Klein
Andrew A
AA
Hicks
Peter P
PP
Pramstaller
Francisco S
FS
Domingues
Irene
I
Pichler
D000818:Animals; D045744:Cell Line, Tumor; D029721:Drosophila Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D004331:Drosophila melanogaster; D006801:Humans; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D044767:Ubiquitin-Protein Ligases; C111567:parkin protein; C487430:park protein, Drosophila