Applying a Novel cADPR Photoaffinity Labelling Analogue to Dissect the Cyclic ADP-Ribose (cADPR)-Ca2+ Signaling in Mammalian Cells

Mobilization of intracellular Ca2+ stores is involved in almost every aspect of many cellular processes. Cyclic adenosine diphosphoribose (cADPR) is an endogenous Ca2+ mobilizing nucleotide present in many cell types and different species, from plants to animals. cADPR is formed by ADP-ribosyl cyclases from nicotinamide adenine dinucleotide (NAD). It has been shown that many extracellular stimuli can induce cADPR production that leads to Ca2+ release or influx, establishing cADPR as a second messenger. Although evidence indicates that cADPR elicits Ca2+ release via ryanodine receptors (RyRs), the molecular mechanisms regarding the cADPR-induced Ca2+ release remain unknown. We hypothesize that novel signaling proteins are required for or can modify the ability of cADPR to induce Ca2+ release. We recently synthesized a fluorescent caged cADPR analogue, coumarin caged isopropylidene protected cIDPRE (Co-i-cIDPRE), and found that it is a potent and controllable cell permeant cADPR analogue. We subsequently performed targeted RNAi screening of 22 known RyRs associated proteins on uncaged Co-i-cIDPRE induced Ca2+ increase in human Jurkat cells. Knockdown of several genes, e.g. CHERP, Calcium homoeostasis modulator 1 (CALHM1), and Homer 1, markedly inhibited uncaged Co-i-cIDPRE induced Ca2+ increase. Yet, the precise roles of these hits in cADPR-Ca2+ signaling remain elusive. Moreover, it is quite possible that other proteins not included in our initial RNAi screening are also involved in cADPR-Ca2+ signaling. Therefore, we chemically synthesized a novel photoaffinity labeling cADPR analogue, PAL-cIDPRE, and found that it is a cell permeant cADPR agonist. In this proposal, we will apply PAL-cIDPRE to (1) dissect the roles and mechanisms of the hits from the RNAi screening, e.g. CHERP, Calsequestrin, and Homer1, in cADPR-mediated Ca2+ mobilization; and (2) identify novel cADPR binding proteins by combining approaches of photoaffinity protein-ligand labeling and mass spectrometric analyses. Given the pivotal role of the cADPR-mediated Ca2+ signaling pathway in a wide variety of cellular processes, understanding the molecular mechanisms involved in this prominent signaling pathway is important not only for scientific reasons but also has clinical relevance.