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

Project: Research

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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.


Project number9042160
Grant typeGRF
Effective start/end date1/11/1418/10/18