In the Myb family, as in other families of transcription factors sharing similar DNA-binding domains (DBDs), diversity of function is believed to rely mainly on the less conserved parts of the proteins and on their distinct patterns of expression. However, small conserved differences between DBDs of individual members could play a role in fine-tuning their function. We have compared the highly conserved DBDs of the three vertebrate Myb proteins (A-, B- and c-Myb) and found distinct functional differences. While A- and c-Myb behaved virtually identically in a variety of DNA-binding assays, B-Myb formed complexes of comparatively lower stability, rapidly dissociating under competitive conditions and showing less tolerance to binding site variations. The three protein domains also differed as substrates for protein kinases. Whereas PKA in theory should target the DBDs of A- and c-Myb, but not B-Myb, only c-Myb was phosphorylated by PKA. CK2 phosphorylated all three proteins, although on different sites in the N-terminal region. Finally, B-Myb was remarkably sensitive to cysteine-directed oxidation compared to the other Myb proteins. Our data suggest that the small differences that have evolved between individual Myb family members lead to clear differences in DBD properties even if their sequence recognition remains the same.

Yeast (Saccharomyces cerevisiae) has proved to be a highly valuable tool in a range of screening methods. We present in this work the design and use of a novel yeast effector-reporter system for selection of sequences recognised by DNA-binding proteins in vivo. A dual HIS3-lacZ reporter under the control of a single randomised response element facilitates both positive growth selection of binding sequences and subsequent quantification of the strength of the selected sequence. A galactose-inducible effector allows discrimination between reporter activation caused by the protein under study and activation due to endogenous factors. The system mimics the physiological gene dosage relationship between transcription factor and target genes in vivo by using a low copy effector plasmid and a high copy reporter plasmid, favouring sequence selectivity. The utility of the novel yeast screening system was demonstrated by using it to refine the definition of an optimal recognition element for the c-Myb transcription factor (MRE). We present screening data supporting an extended MRE consensus closely mimicking known strong response elements and where a sequence of 11 nt influences activity. Novel features include a more strict sequence requirement in the second half-site of the MRE where a T-rich sequence is preferred in vivo.

We found previously that stimulation of natriuretic peptide receptor (NPR)-B by C-type natriuretic peptide (CNP) in failing rat ventricle potentiates β1-adrenoceptor (β1-AR)-mediated inotropic response to noradrenaline through cGMP-mediated inhibition of phosphodiesterase (PDE) 3, thereby enhancing cAMP-mediated signalling. Increased cAMP-mediated signalling is deleterious in chronic heart failure (HF; basis for the use of β-blockers in HF) and we propose to consider NPR-B antagonists as new HF treatment in addition to conventional therapy. Since there is no NPR-B-selective antagonist available for clinical studies, we aimed at identifying a novel small molecule (non-peptide) NPR-B antagonist. An assay was developed and high throughput screening performed on a chemical library of about 20,000 small molecule compounds (<500 Da) to identify NPR-B antagonists based on inhibition of CNP-stimulated cGMP production in NPR-B-expressing HEK293 cells. The screen revealed several potential NPR-B antagonists, of which six were selected for further studies. Three showed selective NPR-B vs NPR-A inhibition and three were partially selective. The compounds mediated reversible, noncompetitive inhibition and most likely act as allosteric modulators binding outside the agonist binding site of NPR-B. In rat ventricular muscle strips, the potentiating effect of CNP upon β1-AR-evoked inotropic effects could be attenuated by at least one of these compounds. We identified several small molecule NPR-B antagonists by high throughput screening and show in a functional heart preparation that blocking NPR-B stimulation with a small molecule compound can reduce the potentiating effect of CNP on the β1-AR-mediated inotropic response to noradrenaline.

The specific binding of a protein to a nucleic acid is a first step in several central processes in a living cell. Sequence‐specific protein–DNA interactions are crucial for the functional read‐out of genetic information. Sequence recognition is the result of a concerted action of many weak chemical interactions of different types between the protein and its DNA target, including nonspecific electrostatic interactions, hydrogen bonding and van der Waals contacts. The precise complementarity of shape between the two macromolecules facilitates specific chemical recognition to be established. The electrophoretic mobility shift assay (EMSA) and several variants of footprinting are simple electrophoretic methods developed to study protein–DNA interactions. Because the specificity is determined by the nucleic acid sequence, the same methods can be exploited for a wide range of proteins simply by changing the sequence of the nucleic acid. EMSA detects sequence‐specific DNA‐binding activity in a protein sample as a separate migrating band in a nondenaturating gel. A footprinting method provides more detailed information on the precise location of a bound protein along the DNA fragment through the removal of specific bands in a pattern of cleaved fragments separated by electrophoresis. Both methods are highly sensitive due to the use of radioactively labeled oligonucleotides and can be performed with protein samples of low purity. When combined these methods are capable of providing a picture of the protein–DNA complex with a great deal of molecular detail, surpassed only by the more demanding methods of crystallography and nuclear magnetic resonance (NMR).