Dissecting the Mechanism and Function of GAPDH in Cyclic ADP-Ribose (cADPR)-mediated Ca2+ Signaling in Mammalian Cells

Project: Research

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The Ca2+-signaling pathway mediated by cyclic adenosine diphosphoribose (cADPR) is ubiquitous and the functions it regulates are equally diverse. Understanding the molecular mechanisms involved in this novel signaling pathway is not only scientifically important but also clinically relevant. The latter has clearly been demonstrated in CD38 knockout mice. CD38 is the dominant enzyme for synthesizing cADPR in mammalian systems and CD38 knockout mice exhibited multiple physiological defects, ranging from impaired immune responses, metabolic disturbances, to social behavioral modifications. Many extracellular stimuli have been shown to induce cADPR production that leads to calcium release or influx, establishing cADPR as a second messenger. Ample evidence indicates that cADPR targets ryanodine receptors (RyRs) on ER in many cell types, yet cADPR does not directly act on the receptor. We hypothesize that a bridging protein is required for the ability of cADPR to induce Ca2+release via RyRs. We, therefore, synthesized a novel photoaffinity labeling cADPR agonist, PAL-cIDPRE, and subsequently applied it to purify its binding proteins in human Jurkat T cells. We identified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of cADPR binding protein(s), characterized the binding affinity between cADPR and GAPDH in vitro by SPR assay, and mapped the cADPR’s binding sites in GAPDH. We further demonstrated that cADPR induced the transient interaction between GAPDH and RyRs in vivo, and GAPDH knockdown abolished cADPR-induced Ca2+release. On the other hand, GAPDH did not catalyze cADPR into any other known or novel compound(s). Our data suggest that GADPH is the long-sought-after cADPR binding protein and is required for cADPR-mediated Ca2+mobilization from ER via RyRs. Based on the simulated cADPR-GAPDH complex structure, we also performed the structure-based drug screening, identified a number of small chemicals with high docking scores to cADPR’s binding pocket in GAPDH, and found one of these compounds, C244, is a potential cADPR antagonist. Here we propose to (1) mechanistically study how cADPR binds with GAPDH to trigger Ca2+release from ER via RyRs, and (2) chemically synthesize and pharmacologically characterize novel cADPR agonist(s) or antagonist(s). Given the pivotal role of cADPR-mediated Ca2+signaling pathway in a wide variety of cellular processes, understanding the molecular mechanisms of this signaling pathways and identifying novel cADPR agonist(s) or antagonist(s) will be fundamentally important for pharmaceutical intervention in treatment of cADPR-related human disorders.?


Project number9042469
Grant typeGRF
Effective start/end date1/01/18 → …