Interactome and Subcellular Analysis of the Tethering Proteins VAPA and VAPB in Cardiomyocytes
by Dana El Chami
Date of Examination:2024-08-20
Date of issue:2025-07-18
Advisor:Prof. Dr. Stephan E. Lehnart
Referee:Prof. Dr. Ralph Kehlenbach
Referee:Prof. Dr. Rubén Fernández-Busnadiego
Referee:Prof. Dr. Ralf Dressel
Referee:PD Dr. Antje Ebert
Referee:Dr. Alexander Stein
Persistent Address:
http://dx.doi.org/10.53846/goediss-11390
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Abstract
English
Heart muscle contraction is regulated by excitation-contraction-coupling (ECC), where calcium (Ca2+) released from the sarcoendoplasmic reticulum (SER) triggers myofilament contraction. The SER is crucial for intracellular Ca2+ cycling in cardiomyocytes, ensuring proper ECC through specialized proteins responsible for Ca2+ release and reuptake. In a previous project, we used a proximity labeling assay coupled with mass-spectrometry-based complexome profiling on murine-isolated ventricular cardiomyocytes (VCMs) to elucidate the interactome of phospholamban (PLN), a key SER protein associated with calcium reuptake. This analysis identified two isoforms of the VAMP-associated protein (VAP) family, VAPA and VAPB, co-migrating with PLN and another key regulator of Ca2+ reuptake, the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA2a). While VAPA and VAPB have known roles in multiple contact site (MCS) formation, Ca2+ homeostasis, lipid transport, and protein proteostasis, their specific functions in ventricular cardiomyocytes remain uninvestigated. Our preliminary data suggest that VAPA and VAPB may regulate Ca2+ homeostasis by interacting with the PLN/SERCA2a complex. In this study, we investigate the role of VAPA and VAPB in VCMs using state-of-the-art techniques, such as co-immunoprecipitation (Co-IP), immunofluorescence coupled with confocal and Stimulated Emission Depletion Microscopy (STED), Ca2+-imaging, and mass spectrometry. We focused on investigating the functions and interactions between VAPA and VAPB and Ca2+ handling proteins in wild-type, PLN knock-out, and VAPB knock-out VCMs. Additionally, we employed a heterologous expression system to elucidate isoform-specific functions since VAPA and VAPB can form homo- and hetero-dimers. Our findings revealed that both VAPA and VAPB are expressed in VCMs and localized to the sarcomeric Z-line alongside SERCA2a. In addition, VAPB and VAPA localized specifically to the M-band and the nuclear envelope, respectively, indicating isoform-specific distributions. Affinity MS revealed potential interacting partners, including previously known tethering proteins, as well as novel interactors. Notably, the novel interactor SERCA2a was detected in the VAPA and VAPB interactome analysis, and the novel interactor small ankyrin 1.5 (sANK1.5) was detected in the VAPB interactome analysis. Co-IP confirmed the interaction between VAPA/VAPB and SERCA2a and PLN, as well as between VAPB and sANK1.5. In VAPB knock-out VCMs, VAPA interacted with SERCA2A and PLN independently of VAPB; however, VAPA was redistributed to the M-band and sANK1.5 to the Z-line. Ca2+ imaging showed enhanced SER Ca2+ load and SERCA2a activity in VAPB knock-out VCMs. Heterologous expression of VAPB, PLN, and SERCA2a in VAPA/VAPB or VAPB knock-out Rabbit Kidney-13 cells demonstrated direct and distinct interactions between VAPB and PLN and between VAPA and SERCA2a. Our findings suggest that VAPA and VAPB act as docking proteins for SERCA2a and PLN, respectively, potentially facilitating PLN inhibition of SERCA2a. Moreover, our data provides the novel insight, that VAPA and VAPB appear to be involved in Ca2+ homeostasis and ECC. Additionally, the mass spectrometry-based interactome analysis confirmed functions in MCS formation and lipid homeostasis in VCMs. Moreover, our results suggest more regulatory proteins and events are involved in the PLN-SERCA2a interaction than previously thought, indicating the presence of fine-tuning proteins that might also serve as potential drug targets for heart disease.
Keywords: Phospholamban; Phospholamban knockout; SERCA2a; Calcium; Heart; Cardiomyocyte; VAPA; superresolution microscopy; VAMP-Associated Protein A; VAMP-Associated Protein B; proteomics; VAPB; STED
