Background: Autophagy is a fundamental cellular rescue pathway against stress that mediates metabolic recycling of various intracellular cargos inside specialized units called autophagosomes. Available evidence suggests benefit of autophagy in rescue of F508del CFTR protein and function (1, 2). Specific autophagy pathways depend on binding of adaptors and receptors with mammalian autophagy gene Atg8, LC3s/GABARAPs subfamily of proteins through a canonical binding motif known as LC3-interacting region (LIR) (3). The N-terminal region directly preceding the core LIR motif often harbors potential phosphorylation sites that stabilize ATG8 binding (4). We recently identified that a cAMP producing enzyme Adenylate Cyclase 6 (AC6) negatively regulates autophagy through inhibition of AMPK-kinase, a key enzyme to influence metabolic stress-induced autophagy (5). Furthermore, AC6 is the primary AC to regulate CFTR activation through cAMP signaling in airway epithelial cells (6).
Methods: Confocal imaging, Fluorescence Resonance Energy Transfer (FRET) Microscopy, Fluorescence Lifetime imaging Microscopy (FLIM), Electrophysiology, Proteomics
Results: In this study, we will determine whether Adenylyl cyclase-associated protein (CAP1), an AC activating protein that harbors two LIR motifs (mined using iLIR Autophagy Database (warwick.ac.uk) (7)), binds LC3, and serves as an autophagy adaptor for AC6 providing a specific regulatory interface for AC6 mediated negative regulation of autophagy. Formation of AC6-CAP1-LC3 inhibits autophagy, and simultaneously uncouples AC6 from its functional interaction with CFTR (Figure 1). We observed that there is a significant upregulation of CAP1 transcript levels in human CF bronchial epithelial cells compared to non-CF cells suggesting a possibility of inhibition of autophagy through an increased AC6-CAP1 assembly in CF cells. In this study, we will test whether knockdown of CAP1 will uncouple AC6 from autophagy, stimulate autophagy and, restore functional association between membrane CFTR and AC6.
Conclusions: This study will help identify one of the causes of reduced autophagy in CF, and a modifying pathway for most efficient rescue of F508del CFTR protein.
Acknowledgements: This work was supported by NIH and CF grants to APN, and Gilead CF scholar award to KA.
References: Luciani A, Villella VR, Esposito S, Gavina M, Russo I, Silano M, et al. Autophagy. 2012;8(11):1657-72.
2. Villella VR, Esposito S, Bruscia EM, Maiuri MC, Raia V, Kroemer G, et al. Front Pharmacol. 2013;4:1.
3. Wirth M, Zhang W, Razi M, Nyoni L, Joshi D, O'Reilly N, et al. Nat Commun. 2019;10(1):2055.
