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Research

Neuroligin

Our laboratory is interested in the structure and function of receptors, enzymes and adhesion molecules involved in neurotransmission. We have been studying functional coupling of receptors to cellular responses, specificity of ligand recognition by nicotinic acetylcholine receptors and acetylcholinesterase, and the structures of cholinesterases, neuroligins and nicotinic receptors. With Barry Sharpless' group at The Scripps Research Institute (TSRI), we have been employed these molecules as templates for "freeze-frame, click chemistry" synthesis of selective agonists and antagonists. Another area of investigation involves the regulation of expression of the genes encoding these proteins during differentiation and synaptogenesis in neurons, skeletal muscle and hematopoietic cells.

Kinetic Studies of Cholinesterase Catalysis

Structure/activity studies, purification and quaternary protein structure of the enzyme acetylcholinesterase (AChE; EC 3.1.1.7) were in focus of early research interests of this laboratory. The first AChE primary structure deduced from the cloned cDNA nucleotide sequence we determined for the enzyme of the fish Torpedo californica in 1986. Since then, we have been using recombinant DNA technology to design and express fish, human and mouse wild type and mutant AChE proteins. We study molecular basis of high catalytic efficiency of AChE, as well as architecture of binding sites for ligands that affect that catalytic activity. Through the use of fluorophores covalently attached to recombinantly engineered cysteines we were able to study flexibility of AChE domains and conformational changes induced by covalent and reversible ligand binding to AChE. Binding kinetics of those ligands is monitored in the millisecond time intervals through the use of stopped-flow technique and conventional fluorimetry and spectrophotometry for longer interaction intervals.

In collaboration with structural biochemists Drs. Yves Bourne and Pascale Marchot from the University of Marseilles, France we revealed through the series of 3D crystal structures, and supporting interaction kinetics nature of interaction of physiological and other substrates and reaction products with the mouse AChE. In the arena of the novel drug design through collaboration with the laboratory of the 2001 Nobel laureate Barry K. Sharpless and the use of their "in situ click chemistry" we succeeded in designing and characterizing extremely potent reversible AChE inhibitors active at the femtomolar concentration scale. Analogous approach is currently being used for the design of novel and more potent reactivators of phosphylated AChE resulting from the exposure to organophosphorous pesticides or warfare nerve agents through collaboration with Sharpless laboratory and laboratory of Dr. Zrinka Kovarik at the Institute for Medical Research and Occupational Health, Zagreb, Croatia.

Autism Candidate Genes Neuroligin and Neurexin: A Proteomic Approach to Structure and Adhesive Function

Synapses are specialized cell junctions responsible for transmission and integration of signaling in the central nervous system. Two families of neuronal proteins, the neurexins and the neuroligins, appear to play a major role in controlling synaptic recognition patterns in the developing brain. Several genetic studies have linked point mutations found in neurexins and neuroligins to autism spectrum disorder and mental retardation, suggesting that loss of neuroligin and or neurexin function during brain development could lead to aberrant behavior and cognitive defects in humans. More broadly, it is becoming apparent that autism's cause may reside in structural or functional abnormalities of synapses.

Using several biophysical techniques (surface plasmon resonance, small angle X-ray and neutron scattering, protein crystallography, and others) as well as cell biological assays based on primary neuronal cultures, our work is primarily aimed at elucidating the three-dimensional structure of the neuroligins and their activity with the neurexins. Information about the structure of the individual components and how they interact, both in vitro and in vivo, is crucial to understand the mechanistic basis of interaction of these extracellular proteins and predict how mutations can affect function of the system.