Final Report Summary - CHEMBIONMR (Using chemical-biology to synthesis and study nuclear receptor proteins) Protein post-translational modifications (PTMs) like phosphorylation are key players in expanding the avenue of translational medicine for heterogeneous diseases like cancer. Despite considerable efforts to understand the relevance of PTMs in the cellular context, we are still in the process of unraveling the complexity of these modifications and their tremendous impact. Nuclear receptors on the other hand are well established drug targets (13% of all medicines target nuclear receptors). Due to their role in disease pathology and cellular homeostasis, the role of PTMs on nuclear receptors are very attractive molecular targets for developing drugs for cancer treatment as well as other chronic diseases. With the constant addition of new PTMs, verification of newly identified proteins changes by traditional methods and correlating the biological significance is a challenging task. We are just beginning to grasp the enormity of the field and its effect on the normal development and disease patho-physiology. Continued search and evaluation of various functional modifications of nuclear receptors and understanding their interaction in various biological pathways will have important implications in the successful development of novel prognostic markers as well as therapeutic targets for cancer. A limiting factor so far in PTMs and breast cancer research has been the lack of appropriate synthetic approaches for well-defined Estrogen Receptor (ER) constructs featuring PTMs. To overcome this problem, our aim in this research was to use an efficient protein semi-synthetic approach that combined: a) the chemical synthesis of peptides (< 50 amino acids) that contain the desired modifications, b) the biological expression of large sections (> 100 amino acids) of the ER, and c) a final ligation step between the two segments. This strategy allowed the introduction of PTMs at the C-terminus of ER Ligand Binding Domain, in combination with a site-specifically introduced fluorescent probe or 15N-labeled amino acids. Several biophysical techniques (FP, CD and NMR) in combination with molecular dynamics calculations allowed to directly probe the effect of these modifications on the ER dynamics and its role in modulating the ER – coactivator binding. The results reveal the molecular basis for a phosphorylation dependent and ligand-independent pathway for ER activation. Moreover, our data provides evidence for a subtype specific ER activity regulation mechanism. As conclusion, a novel mechanism of ligand-independent activation of ER can be proposed, induced by the phosphorylation of conserved tyrosine residues.The long-term goal of this project would be to reach a good level of understanding of the role of PTMs in processes such as drug action and resistance in ER. The knowledge gained at the molecular level, in this direction, would be of high importance for advancing prevention, early detection and monitoring of the breast cancer disease, and moreover, could guide and promote new therapeutic strategies for breast cancer treatment.