Abstract
Myocyte enhancer factor 2 (MEF2) proteins play a pivotal role in the differentiation of cardiac and skeletal muscle cells. MEF2 factors are regulated by histone deacetylase enzymes such as histone deacetylase 5 (HDAC5). HDAC5 in turn is responsive to Ca(2+) signaling mediated by the intracellular calcium sensor calmodulin. Here a combination of proteolytic fragmentation, matrix-assisted laser desorption ionization mass spectrometry, Edman degradation, circular dichroism, gel filtration, and surface plasmon resonance studies is utilized to define and characterize a stable core domain of HDAC5 and to examine its interactions with MEF2a and calmodulin. Results from real time binding experiments provide evidence for direct interaction of Ca(2+)/calmodulin with HDAC5 inhibiting MEF2a association with this enzyme.
Highlights
In eukaryotes, transcription occurs in the nucleus where the DNA template is packaged in chromatin
Direct interaction of Ca2ϩ/CaM was demonstrated to inhibit histone deacetylase 5 (HDAC5) repressor core binding to MEF2a
Myocyte enhancer factor 2 (MEF2)-controlled myogenic genes that are active in early myocyte development need to be efficiently repressed in the adult muscle cell
Summary
Protein Expression and Purification—Human HDAC5 repressor core polypeptides were expressed as fusion proteins containing a His tag at the C terminus. All expressed proteins (RprcL, RprcS, RprcS(L187G), CaM, and MEF2a) were purified to homogeneity as verified by Coomassie staining, N-terminal sequencing, and mass spectrometric analysis. The sensor chip was regenerated with 50 l of Buffer M containing 500 mM NaCl. Binding experiments of HDAC5 RprcL protein to MEF2a were carried out in Buffer R1 (25 mM BisTris, 100 mM NaCl, 0.5 mM EDTA, 1 mM DTT, pH 7.0) with protein concentrations ranging from 2 to 35 nM. Binding experiments of RprcS to MEF2a were carried out at pH 6.2 For both RprcL and RprcS, it was found that at the concentrations utilized, the best reproducibility of data was achieved by diluting protein solutions from a 1 M stock with running buffer immediately prior to injection.
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