Proximity Labeling-Based Network Analysis to Probe Molecular Functions of Adenylyl Cyclase-Containing Macromolecular Complexes in Cardiomyocytes

Abstract

<b>Abstract ID 23771</b> <b>Poster Board 483</b> The second messenger cAMP is essential for sympathetic and parasympathetic regulation of cardiomyocytes. The spatial organization of cAMP signaling is key to the unique functional responses elicited by different hormonal inputs. This organization is achieved in part by means of scaffolding proteins that coordinate adenylyl cyclase (AC) localization in conjunction with downstream effectors of cAMP pathway, thus sensitizing the system to low levels of cAMP. However, the composition and dynamics of AC-containing macromolecular complexes in heart disease remain mostly unknown, particularly for different AC isoforms. We have carried out a comprehensive proteomic approach of AC complexes in the heart using BioID (proximity-dependent identification of near-neighbor interaction). We have fused the mini-Turbo biotin ligase with three different cardiac AC isoforms and control GFP and have expressed these fusion proteins in neonatal cardiomyocytes using adenoviruses. Neonatal cardiomyocytes were subsequently incubated with biotin for 3 hours and harvested for streptavidin pull-down and mass spectrometry. Proteins identified by BioID were incorporated into Significance Analysis of INTeractome (SAINT) and high-confidence prey proteins were selected by Saint Score (0.7 and above). High-confidence prey proteins have been classified with their enriched Gene Ontology terms (GO) and cellular compartments using gProfiler, SAINT, and UniProtKB. Distribution of interactomes represented unique protein patterns for different AC isoforms, particularly with respect to GO localization terms and cellular responses to stress. To confirm our screening results, we performed Flag-pull down mass spectrometry with different AC isoforms and compared the derived interactomes with those from BioID. Proteins identified by Flag-MS overlapped with BioID-derived interactors (35-60%, depending on the AC isoform), with similar enrichment in GO terms relating to protein folding, localization, and small GTPase binding function. We further validated one specific prey protein for AC5 and AC6 that was identified by BioID and Flag-MS - an E3 ubiquitin ligase, STUB1 (also called CHIP). STUB1 interaction with AC5/6 has been assessed by IP-Western blot analysis, immunocytochemistry, and its functional consequences on AC activity verified by knock-down siRNA experiments. Our current comprehensive network analysis of complex formation is expected to provide important insights for understanding the spatial and functional significance of AC-containing macromolecular complexes in the heart. This research was funded by National Institute of General Medical Science RO1GM060419 and the Mathers Foundation