ISSN: 2577-4360
Authors: Liu M, Zaman R, Jafri A, Holloway K, Hussain F, Knight J, Sawczak V, Getsy P, Cao R, Raffay T, Sun F, Cotton C, Periasamy A, Zaman K* and Lewis SJ
Introduction: Cystic fibrosis (CF) is a multisystem disease associated with mutations in the gene that encodes the CF transmembrane conductance regulatory (CFTR) protein. S-nitrosothiols (SNOs) are endogenous cell signaling molecules with relevance to human lung disease, including cystic fibrosis (CF). S-nitrosoglutathione (GSNO), one class of SNOs levels are significantly lower in the CF airway than the healthy airway. Here, we address whether GSNO interact with activator of the Hsp90 ATPase (Aha1), which targets improperly folded CFTR for degradation. Materials and methods: We used human bronchial airway epithelial (CFBE410-) cells expressing wild type and mutant F508del CFTR protein. Immunoblot analysis was performed in the presence or absence of GSNO by using monoclonal antibodies for Aha1 and CFTR. We knocked down endogenous Aha1 with Aha1 siRNA duplexes. Next, we tested Aha1 S-nitrosylation by using a biotin switch assay and measurement of ATPase activity by using the EnzChek phosphate detection kit. Finally, we used confocal laser scanning microscopy for co-localization of Aha1 and CFTR. Results: Our study showed that GSNO significantly decreased Aha1 expression. Following Aha1 knockdown, CFTR maturation levels were elevated at the cell surface in F508del CFTR. In the presence of GSNO. F508del CFTR maturation was further increased. We found that CFTR-associated Aha1 is S-nitrosylated and inhibits its ATPase activity by GSNO. Finally, we showed cellular co-localization of Aha1 with CFTR by confocal microscopy. Conclusion: Our observations offer a novel approach in identifying the key mechanism by which SNOs increase F508del CFTR maturation and cell membrane stabilization.
Keywords: Cystic fibrosis; CFTR; S-nitrosoglutathione; Chaperones; Aha1
