Molecular mechanisms underlying activation and regulation of ADP-ribosyl cyclases and their functional role in generating cADPR and NAADP. (360G-Wellcome-079254_Z_06_Z)

Intracellular calcium mobilizing messengers cyclic ADP-ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mediate many important physiological processes. Both cADPR and NAADP are believed to be generated by ADP-ribosyl cyclase (ARC) enzymes. However, information on how these enzymes are regulated by extracellular stimuli is almost completely lacking. Sea urchin egg homogenates represent a robust cell-free calcium release assay for biochemical reconstitution of endogenous signalling pathways. In this project we will characterize recently identified sea urchin ARC(s), enabling detailed biochemical dissection of ARC regulation and identification of ARC-associated proteins that mediate signalling cascades regulating ARC activity. By generating molecular tools that activate or inhibit ARC signalling components, we will identify ARCs' functional role during sea urchin fertilization and embryogenesis and gain general key insights into their mode of regulation. A central goal in this respect is to extend our findings to identify how mammalian ARCs are regulated during important physiological processes. We will identify those proteins that interact with mammalian ARCs in exocrine pancreatic cells and thus uncover the signalling pathways regulating them. We will also use a variety of approaches to functionally inhibit ARCs in these cells and thus uncover their functional role in the exocrine pancreas.