Phosphorylation-dependent Interactome of Ryanodine Recep-tor Type-2 in the Heart

ABSTRACT: Background: Hyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrilla-tion and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 -S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 in-teractome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the un-phosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins with 20 being high confident RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blot and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.

INSTRUMENT(S): LTQ Orbitap Discovery

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Heart

SUBMITTER: David Chiang  

LAB HEAD: Xander Wehrens

PROVIDER: PXD026350 | Pride | 2021-06-07

REPOSITORIES: Pride