Abstract
Purpose :
Mutations in ARL2BP (ADP-ribosylation factor-like 2 binding protein) are associated with ciliopathies in humans, causing retinitis pigmentosa, situs inversus, and male infertility. Our studies using Arl2bp KO mouse model revealed that ARL2BP is necessary for photoreceptor ciliary doublet microtubule formation and axoneme elongation, which is required for the morphogenesis of photoreceptor outer segments. However, the molecular mechanisms underlying the ciliopathies caused by ARL2BP mutations are not fully understood. We hypothesize that ARL2BP mutations result in aberrant interaction with its binding proteins, leading to structural defects in cilia and blindness. Therefore, the goal of this study is to identify novel ARL2BP interacting partners.
Methods :
Co-immunoprecipitation (Co-IP) of ARL2BP with its binding proteins, followed by Mass Spectrometry, was utilized to identify interacting partners of ARL2BP in the retina and testes. ARL2BP protein-protein interaction was confirmed by exogenous expression in HEK293T cells followed by Co-IP.
Results :
Co-IP of ARL2BP followed by Mass Spectrometry identified CFAP20 (Cilia- and Flagella-Associated Protein 20) as one of the potential interacting partners of ARL2BP. This protein-protein interaction was confirmed by exogenous expression of HA-tagged ARL2BP and FLAG-tagged CFAP20. CFAP20 was Co-IPed with HA-IP, and ARL2BP was co-IPed with FLAG-IP, thus confirming ARL2BP-CFAP20 interaction. Interestingly, co-expression of ARL2BP-HA and CFAP20-FLAG resulted in 6- and 2.5-fold increase in ARL2BP-HA and CFAP20-FLAG, respectively, suggesting the importance of the ARL2BP-CFAP20 interaction for the stability of these proteins.
Conclusions :
This study identified and confirmed CFAP20 as one of the interacting partners of ARL2BP. Interestingly, a recent study linked mutations in human CFAP20 with retinitis pigmentosa and fertility issues, phenotypes found in ARL2BP patients. Moreover, lack of CFAP20 in C.elegans lead to open ciliary doublet microtubules, structural defects that are also observed in ARL2BP null murine photoreceptors cilia. Altogether, these findings suggest an important role for ARL2BP-CFAP20 interaction in the structural integrity of cilia. Our current efforts are focused on understanding the relevance of ARL2BP – CFAP20 interaction and its function in cilia.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.