Dissecting the Mechanism and Function of GAPDH in Cyclic ADP-Ribose (cADPR)-mediated Ca2+ Signaling in Mammalian Cells
DescriptionThe Ca2+-signaling pathway mediated by cyclic adenosine diphosphoribose (cADPR) isubiquitous and the functions it regulates are equally diverse. Understanding themolecular mechanisms involved in this novel signaling pathway is not only scientificallyimportant but also clinically relevant. The latter has clearly been demonstrated in CD38knockout mice. CD38 is the dominant enzyme for synthesizing cADPR in mammaliansystems and CD38 knockout mice exhibited multiple physiological defects, ranging fromimpaired immune responses, metabolic disturbances, to social behavioral modifications.Many extracellular stimuli have been shown to induce cADPR production that leads tocalcium release or influx, establishing cADPR as a second messenger. Ample evidenceindicates that cADPR targets ryanodine receptors (RyRs) on ER in many cell types, yetcADPR does not directly act on the receptor. We hypothesize that a bridging protein isrequired for the ability of cADPR to induce Ca2+release via RyRs. We, therefore,synthesized a novel photoaffinity labeling cADPR agonist, PAL-cIDPRE, andsubsequently applied it to purify its binding proteins in human Jurkat T cells. Weidentified glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as one of cADPRbinding protein(s), characterized the binding affinity between cADPR and GAPDH invitro by SPR assay, and mapped the cADPR’s binding sites in GAPDH. We furtherdemonstrated that cADPR induced the transient interaction between GAPDH and RyRsin vivo, and GAPDH knockdown abolished cADPR-induced Ca2+release. On the otherhand, GAPDH did not catalyze cADPR into any other known or novel compound(s). Ourdata suggest that GADPH is the long-sought-after cADPR binding protein and isrequired for cADPR-mediated Ca2+mobilization from ER via RyRs. Based on thesimulated cADPR-GAPDH complex structure, we also performed the structure-baseddrug screening, identified a number of small chemicals with high docking scores tocADPR’s binding pocket in GAPDH, and found one of these compounds, C244, is apotential cADPR antagonist. Here we propose to (1) mechanistically study how cADPRbinds with GAPDH to trigger Ca2+release from ER via RyRs, and (2) chemicallysynthesize and pharmacologically characterize novel cADPR agonist(s) or antagonist(s).Given the pivotal role of cADPR-mediated Ca2+signaling pathway in a wide variety ofcellular processes, understanding the molecular mechanisms of this signaling pathwaysand identifying novel cADPR agonist(s) or antagonist(s) will be fundamentally importantfor pharmaceutical intervention in treatment of cADPR-related human disorders.?
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