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Abstract:
An in vitro screening method was developed which allows to identify proteinprotein-interactions on protein arrays by overlaying with a radiolabeled protein probe. First, various PCR products were generated which code for proteins that are believed to play a role in neurodegenerative disorders. Among them were several proteins which have been reported previously to interact with each other. In a proof-of-principle approach 96 His-tagged proteins were immobilized on a PVDF membrane and were assayed for their interaction with radiolabeled GST fusion protein of the C-terminus of 70 kDa heat shock cognate protein (Hsc70), the full length heat shock organizing protein (HOP) and the SH3 domain of endophilin-1 (SH3E1). Interaction of Hsc70 with HOP as well as SH3E1 with amphiphysin-1 was demonstrated in this proof-of-principle approach. To discover novel interaction partners for the SH3E1, the developed screening method was extended to a genome-wide scale using high-density protein arrays from a human fetal brain cDNA expression library. The high-density protein array consisted of 36,864 individual clones spotted in duplicates by a robot on two PVDF membranes. Robotic spotting was performed at the German Resource Center RZPD. To distinguish false positive signals, a second high-density protein array was overlayed with 60 kDa heat shock protein (p60) as a negative control. The screening approach identified 30 clones from the cDNA library which expressed a protein in frame with the vector-encoded Histag and were specific for SH3E1. One clone expressed a C-terminal fragment of (ALG-2)-interacting protein 1 (Alix), a previously reported interaction partner of endophilin-1. His-tagged proteins were purified on NiNTA agarose beads and used in a pulldown assay to confirm the binding of SH3E1 to the discovered interaction partners in vitro. Possible binding regions of SH3E1 were mapped, using scans of peptides delineated from the identified interaction partners. Combination of the overlay screen with independent
pulldowns and peptide scans confirmed the interaction of SH3E1 with Alix, disks largeassociated protein 4 (DAP4), cysteine and glycine-rich protein 2 (CRP2), capicua
homolog (CIC), heterogenous nuclear ribonucleoprotein A1 (HNRPA1), microtubuleassociated protein 1A light chain 3A (MAP1ALC3), SLIT-ROBO Rho GTPase activating protein 1 (SRGAP1), Gli-Krüppel family member (GLI3) and the hypothetical proteins FLJ10101, C18ORF11 and KIAA1295 and mapped possible binding regions for Alix, DAP4, CIC, MAP1LC3A, SRGAP1, FLJ10101, C18ORF11, KIAA1295 and NGFI-A
binding protein 2 (NAB2).
