Non-erythroid beta spectrin interacting proteins and their effects on spectrin tetramerization

• Published in: Cellular & molecular biology letters 2011-12, Vol.16 (4), p.595-609
• Main Authors: Sevinc, Akin, Fung, Leslie
• Format: Article
• Language: English
• Subjects: Binding Sites; Brain - cytology; Brain - metabolism; Brain beta spectrin; Brain proteins; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell Line; Cytoskeleton - chemistry; Cytoskeleton - metabolism; Gene Library; Humans; Library screening; Microfilament Proteins - chemistry; Microfilament Proteins - genetics; Microfilament Proteins - metabolism; Models, Molecular; Neurons - cytology; Neurons - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Plasmids; Polymerization; Protein Binding; Protein Isoforms - chemistry; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein Structure, Tertiary; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrin interacting proteins; Spectrin tetramerization; Transfection; Two-Hybrid System Techniques; Yeast three-hybrid
• ISSN: 1689-1392; 1425-8153; 1689-1392
• DOI: 10.2478/s11658-011-0025-9

With yeast two-hybrid methods, we used a C-terminal fragment (residues 1697–2145) of non-erythroid beta spectrin (βII-C), including the region involved in the association with alpha spectrin to form tetramers, as the bait to screen a human brain cDNA library to identify proteins interacting with βII-C. We applied stringent selection steps to eliminate false positives and identified 17 proteins that interacted with βII-C (IPβII-C s). The proteins include a fragment (residues 38–284) of “THAP domain containing, apoptosis associated protein 3, isoform CRA g”, “glioma tumor suppressor candidate region gene 2” (residues 1-478), a fragment (residues 74–442) of septin 8 isoform c, a fragment (residues 704–953) of “coatomer protein complex, subunit beta 1, a fragment (residues 146–614) of zinc-finger protein 251, and a fragment (residues 284–435) of syntaxin binding protein 1. We used yeast three-hybrid system to determine the effects of these βII-C interacting proteins as well as of 7 proteins previously identified to interact with the tetramerization region of non-erythroid alpha spectrin (IPαII-N s) [1] on spectrin tetramer formation. The results showed that 3 IPβII-C s were able to bind βII-C even in the presence of αII-N, and 4 IPαII-N s were able to bind αII-N in the presence of βII-C. We also found that the syntaxin binding protein 1 fragment abolished αII-N and βII-C interaction, suggesting that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.