Non-erythroid beta spectrin: Effects of mutations and of interacting proteins on tetramerization
Başlık çevirisi mevcut değil.
- Tez No: 402027
- Danışmanlar: DR. LESLIE WO-MEI FUNG
- Tez Türü: Doktora
- Konular: Biyokimya, Kimya, Biochemistry, Chemistry
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2011
- Dil: İngilizce
- Üniversite: University of Illinois at Chicago
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 145
Özet
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Özet (Çeviri)
Spectrin performs its fundamental roles by forming a filamentous network beneath the plasma membrane, where the tetramerization of the spectrin heterodimers is crucial. We employed yeast two-hybrid (Y2H) methods to study the mutational effect of non-erythroid alpha spectrin (αII) at position 22 in tetramer formation with non-erythroid (beta) spectrin (βII), and to screen a human brain cDNA library to identify proteins interacting with βII-C, using a Cterminal fragment (residues 1697-2145) of non-erythroid beta spectrin (βII-C) as the bait. This region includes the tetramerization region involved in the association with alpha spectrin. For the first part, interaction of wild type (αII-N) and 4 mutants (αII-N-V22D, -V22F, -V22M, and -V22W) of the first 359 residues at the N-terminal region with βII-C, were studied using colony growth and β-galactosidase activity assays of Y2H analysis simultaneously with isothermal titration calorimetry (ITC) analysis. Y2H results showed that the Cterminal region of βII interacts with the N-terminal region of αII, either the wild type, or those with V22F, V22M or V22W mutations. The V22D mutant did not interact with βII. For the positive results, we were not able to detect any differences in interactions between V22F, V22M or V22W with βII-C. Both colony growth rate and colony size, as well as the blue color indication for β-galactosidase activity did not show detectable differences between V22, V22F, V22M and V22W. ITC results showed that the Kd values for V22F were similar to those for the wild-type (about 7 nM), whereas the Kd values were about 35 nM for V22M and about 90 nM for V22W. We were not able to detect any binding for V22D with ITC methods. This study clearly demonstrates that the single mutation at position 22 of αII, a region critical to the function of non-erythroid α spectrin, may lead to a reduced level of spectrin tetramers and abnormal spectrin-based membrane skeleton. These abnormalities could cause abnormal neural activities in cells. For the second part, we identified 17 proteins that interacted with βII-C (IPβII-C s). The interacting 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 zincfinger 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) (Oh and Fung, 2007) 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. This suggests that this protein may inhibit or regulate non-erythroid spectrin tetramer formation.
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