| dc.description.abstracteng | Cardiac contraction and relaxation depend on intracellular Ca2+ release and sequestration in the sarcoplasmic reticulum (SR). The small tail-anchored protein phospholamban (PLN) is essential for normal stress-adaptation of heart function. The dephosphorylated PLN form binds to the Ca2+ ATPase SERCA2a and potentially to other important SER proteins and thereby regulates the reuptake of Ca2+ into the SR. To elucidate the PLN associated signalosome in endogenous SR domains, we developed a PLN-specific proximity assay and applied complexome profiling on native cardiac membranes.
Here, a mass spectrometry-based method described as complexome profiling, originally established to identify OXPHOS protein supercomplexes and assembly factors, is used to elucidate the composition of essential protein complexes important for the intracellular Ca2+ cycling in ventricular cardiomyocytes. Digitonin solubilized, enriched membrane fractions of isolated ventricular cardiomyocytes from wildtype and phospholamban knockout mice were loaded and separated by blue native gradient gel electrophoresis (BN-PAGE). Gel lanes were cut, and trypsin digested followed by mass spectrometry (LC-MS/MS).
Hierarchical clustering and analysis of migration patterns confirmed distinct groups of co-migrating proteins, most prominently OXPHOS complexes, which were used for quantitative validation. Importantly, at higher molecular weight, a novel SR Ca2+ cycling complex comprised of the RyR2 calcium release channel, SERCA2a, and each regulatory protein subunits were identified. Furthermore, the sarcolemmal membrane-associated protein (SLMAP) was identified as a potential interacting protein of PLN.
Combined with phospholamban knockout, complexome profiling enabled the close-to-native analysis of previously unknown macromolecular protein complexes comprised of dual RyR2-SERCA2a transporters, changes in abundance due to phospholamban deficiency.
Additionally, for proteomic mapping, APEX2, a genetically engineered peroxidase, was fused N-terminally to generate APEX2-PLN. Adenoviral expression and biotin-phenol treatment were used to label proteins in nanometric proximity of PLN by biotinylation in living neonatal rat cardiomyocytes (NRCMs). APEX2-biotinylated proteins were enriched by pulldown, processed, and analyzed by mass spectrometry (LC-MS/MS). Additionally, for ratiometric analysis, NRCMs were cultivated with stable isotope labeled amino acids (SILAC). A truncated APEX2-PLNΔ(1-29) construct was used as SR-targeted control and enhanced green fluorescent protein (eGFP) as a negative control.
APEX2-PLN expression in NRCMs was confirmed by Western blot (WB) and fluorescence microscopy through the bicistronically expressed eGFP. Confocal analysis showed that APEX2-PLN co-localizes with endogenous SERCA2a similar to endogenous PLN. The medium and heavy labeled amino acids (SILAC) were incorporated at a rate of >95%, thus enabling global quantitative proteomic analysis. 14-3-3 proteins were identified as significantly enriched gene family for APEX2-PLN biotinylation.
A PLN-specific strategy for proximity labeling was successfully developed in live NRCM and verified for known interaction partners. Proteomic proximity analysis identified previously unknown PLN protein-protein interactions in the neonatal heart. Furthermore, these potential interactors will be exploited in the adult heart, i.e. by combining the proximity data with high-resolution microscopy. | de |