Splicing pattern of Aryl hydrocarbon interacting protein like 1 (AIPL1) in relation to Centromere Protein F

Abstract

Purpose: The photoreceptor specific chaperone AIPL1 is one of the 23 proteins whose genes are involved in the etiology of early-onset severe retinal dystrophies (EOSRD). AIPL1 has been shown to underlie a very severe form of EOSRD, which is attributed to its chaperone function on NUB1 and PDE6A. PDE6A-associated retinal degeneration is much less severe in progression than EOSRD and a NUB1-associated retinal phenotype is unknown. To understand the severity of the AIPL1-associated phenotype we thought to identify other possible partners for AIPL1 that imply a more serve phenotype upon disrupted interaction. One of the clones isolated in a yeast-2-hybrid (Y2H) assay with a photoreceptor specific cDNA-library contained the C-terminus of centromere-specific protein F gene (CENPF). Here a possible interaction of AIPL1 and CENP-F was evaluated.Methods: All splice variants of AIPL1 were cloned into pQE-Tris system with C-terminus His-tag. Over expression studies were carried out followed by SDS-PAGE and Westernblot analysis. Purification of expressed AIPL1 is carried out using Ni-NTA coloumns and purified proteins were detected using the AIPL1 Ab and His Ab. HEK293 and HeLa cells were seeded in silicone frames on microscope slides and grown at 37°C and 5% CO2 over night in an incubator. The constructs were transfected and expressed in HEK293 and HeLa cells. His-tagged AIPL1 was detected by Anti-His antibody (ab) (ab1187, Abcam) and anti-AIPL1-ab (Chicken IgY against purified whole protein). CENP-F was detected by anti-CENPF-ab (sc-135865, Santa Cruz). Immunoreactivity (IR) was recorded on a Keyence (BZ-8100E) wide-field microscope by epifluorescence using the Z-stack feature.Results: Westernblot analysis confirmed protein expression and purification strategy. IR against the AIPL1-specific ab and the His-tag-specific ab for AIPL1 was seen in the cytoplasm of HEK293 cells. IR to intrinsically expressed CENP-F also localized to the cytoplasm in HEK293 cells overlapping with the IR of AIPL1.Conclusions: AIPL1 has chaperone function on an as of yet incompletely elucidated set of proteins. An Y2H assay indicated an interaction of AIPL1 and CENP-F and co-localization inside HEK293 cells provides further support for this finding. Whether AIPL1 supports CENP-F function or CENP-F is need for AIPL1 function is unclear at this time. With CENP-F a partner in the housekeeping pathways of the cell is identified that supports a severe and highly progressive phenotype seen in AIPL1 patients by its activity as microtubule organizer. Question lies ahead is what could be the role of AIPL1 in stabilization of rods and cones in relation to CENP-F cell cycle progression.