STRUCTURAL CHARACTERIZATION AND TRANSCRIPTIONAL REGULATION OF THE CYTOSOLIC PSD-95 INTERACTING PROTEIN (CYPIN) AND ITS ROLE IN NEURONAL DENDRITE BRANCHING By

Dendrite morphology regulates how a postsynaptic neuron receives information from presynaptic neurons. The specific patterning of dendrite branches is promoted by extrinsic and intrinsic factors that trigger the activation of functional signaling pathways. However, only a handful of the regulatory factors and biochemical mechanisms involved in determining dendrite morphology are known. The Firestein laboratory previously reported that cypin (cytosolic PSD-95 interactor), the mammalian guanine deaminase (GDA), plays an active role in regulating dendrite branching in hippocampal neurons. Cypin-promoted increases in dendrite number are dependent on binding of zinc ions to cypin and cypin's guanine deaminase activity. This work focuses on the identification of specific structural properties of cypin that lead to the multifunctional roles in guanine metabolism and dendrite iii development. We first employed phylogenetic analysis and computational structure modeling techniques to construct a three dimensional structural model of cypin. Inspection of our structural model confirmed that specific predicted residues coordinate with a zinc ion to play a role in enzymatic activity and promotion of dendrite branching in developing neurons. In addition, we used a combination of protein structure analysis, experimental kinetic studies, and cell culture tests to uncover novel potential ligands for cypin. We obtained a list of compounds that demonstrate higher binding affinity to GDA than does guanine. Our results provide evidence that an in silico drug discovery strategy coupled with in vitro verification can be successfully implemented to discover compounds that may have therapeutic value for the treatment of diseases and disorders where GDA activity is abnormal. Since the regulation of dendrite branching function by cypin is dependent on intracellular levels of cypin protein, idenitification of transcriptional regulators of cypin gene expression may elucidate how cypin regulates neurite development. Therefore, we also investigated how extracellular factors can regulate cypin expression. Our data show that in developing neurons, BDNF increases cypin protein via activation of the MEK pathway, and consequently, CREB transcription factor-dependent cypin gene expression. The discovery of intrinsic regulators of cypin expression aids in our understanding of molecular mechanisms underlying dendritic patterning, and hence, synaptic plasticity, learning and memory.