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V. P. Theendakara, N. B. Akhmedov, C. Yamashita, M. Saghizadeh, D. B. Farber; The Role of the ZBED4 Protein in Cone Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3420.
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© ARVO (1962-2015); The Authors (2016-present)
ZBED4 is a novel protein expressed in cone photoreceptors and glial Müller cells. At the subcellular level, ZBED4 is expressed both in the nucleus and cytoplasm of the human retina. The predicted protein has four zinc BED-finger domains that are distinct from the classic C2H2 Zn-fingers, two nuclear receptor-interacting modules (LXXLL), and a hATC dimerization domain. LXXLL modules are known to bind to a hydrophobic cleft in nuclear hormone receptors (NHRs) resulting from a ligand-dependent conformational change, leading to remodeling of chromatin structure, recruitment of RNA polymerase II, and transcriptional activation. Our purpose is to study whether ZBED4 associates directly with ligand-bound NHRs or if it acts in vivo either as part of a co-activator/co-repressor complex or downstream of a co-activator/co-repressor complex to modulate transcriptional activity.
HEK293 cells expressing ZBED4 were lysed in RIPA buffer containing protease inhibitors. The lysate was reacted with protein G-agarose beads and then incubated with the ZBED4 antibody. After pelleting and washing the beads, bound proteins were eluted in SDS sample buffer. The immunoprecipitated proteins were then separated on a 4-20% Tris-glycine gel and stained with SYPRO-Ruby. The protein bands were cut and processed for mass spectroscopy.
ZBED4 and MYH9 (non-muscle type II myosin heavy chain) were among the 6 proteins identified by mass spectroscopy. To corroborate these results, the immunoprecipitated proteins were immunoblotted with anti-ZBED4 and anti-MYH9 antibodies. The presence of both ZBED4 (135kDa) and MYH9 (224kDa) proteins on the immunoblots confirmed the interaction of ZBED4 with MYH9. Non-muscle myosin heavy chain 9 (MYH9), an actin-based motor protein, has been identified as a physical linker between nucleolin and the cytoskeleton, thus modulating the translocation of nucleolin.
Our preliminary results indicate an interaction of ZBED4 with MYH9. We are currently investigating the co-localization of these proteins by immunocytochemistry and determining whether MYH9 is involved in the translocation of ZBED4 between the nucleus and the cytoplasm.
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