Molecular and Functional Characterisation of Sea Urchin ADP-ribosyl Cyclases

All cells of the body are physically separated from their surrounding environment by a membrane. In order for cells to respond to factors in the blood (such as hormones) which normally can not pass through the membrane, special 'messenger' molecules are used that are generated inside the cell upon contact of the stimulus with the cell. Two of these messengers called cADPR and NAADP are produced by a single family of enzymes / the ADP-ribosyl cyclases. Once made, cADPR and NAADP cause large increases in the concentration of calcium within the cell which in turn are important for many processes including for example the nerve function. Understanding how these enzymes work then is important to understand how cells work. In this study we will determine the DNA sequences of the genes for ADP-ribosyl cyclases in the sea urchin a well-studied experimental system. Having this information will allow us to produce large quantities of each protein to study their properties and determine their location within eggs. We will then study how these enzymes change when eggs are activated by sperm and determine what impact blocking these enzymes has on how the embryo develops. This study should provide new information on these critically important proteins.

ADP-ribosyl cyclases constitute a remarkable family of enzymes capable of catalyzing multiple reactions including the synthesis of the novel and potent Ca2+ mobilizing messengers, cADPR and NAADP. These enzymes are thus likely to play central roles in Ca2+ signaling - a process vitally important for a myriad of cellular events. Not all ADP-ribosyl cyclases however have been characterized at the molecular level. Furthermore, although both cADPR and NAADP have been implicated in fertilization their role during subsequent development is not known. The objective of this project is to provide a complete molecular description of ADP-ribosyl cyclases in a model organism (the sea urchin) and to define the functional role of these enzymes during early development. Building on substantial preliminary data, we will apply molecular and proteomic approaches to isolate sea urchin ADP-ribosyl cyclases and characterize their catalytic properties. Using molecular and immunological assays, we will define the complement of sea urchin ADP-ribosyl cyclases in eggs and the developing embryo and perform parallel biochemical measurements of activity and messenger levels. By inhibiting ADP-ribosyl cyclase expression, we will establish their role in i) the generation in vivo of cADPR and importantly, NAADP (of which little is known) and ii) the course of development. This study will provide novel insight in to the action of these highly versatile multifunctional enzymes.