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Ideke J.C. Lamers, Filine van den Bosch, Kim Klarenaar, Lisa Elze, Marius Ueffing, Karsten Boldt, Ronald Roepman; Dissecting the function of IQCB1/NPHP5, associated with retinal ciliopathies, by targeting its endogenous interactome. Invest. Ophthalmol. Vis. Sci. 2020;61(7):4008.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in IQCB1/NPHP5 cause defects in photoreceptor sensory cilia, resulting in syndromic and non-syndromic Retinitis Pigmentosa and Leber Congenital Amaurosis. Although several proteins were found to interact with IQCB1, these affinity studies were likely compromised by IQCB1 overexpression that could dramatically affect protein complex stoichiometry, structure, and function. This conceptual limitation has rendered a detailed function of IQCB1 elusive to date. We hypothesize that affinity proteomics of epitope tagged endogenous IQCB1, would yield more accurate data on the IQCB1 protein complex constitution, relevant to understanding the associated disease mechanisms and therapeutic options.
In this study, we deployed CRISPR-Cas9 mediated Homology Directed Repair (HDR) of the IQCB1 locus, to fuse it in-frame with a fragment encoding the miniTurboID proximity labeling tag. This enabled proximity proteomics followed by mass spectrometry, as well as epitope tag-directed immunocytochemistry in HEK293T cells. Comparison to several Tandem Affinity Proteomics (TAP) datasets of both transiently and stably expressed IQCB1 pinpoint the specific IQCB1 interactome and demonstrate the power of this approach.
TAP followed by mass spectrometry of epitope tagged IQCB1 in transiently transfected HEK293T cells, or upon stable overexpression in Flp-in HEK293T cells, identified interactomes of 174 and 109 proteins, respectively, of which 72 proteins overlap. Strikingly, the detection yield of the bona fide interactor of IQCB1, CEP290, was raised 7-fold upon stable IQCB1 expression. The endogenously expressed miniTurboID-tagged IQCB1 protein shows a considerably lower expression level and exclusive localization at the centrosomes and ciliary basal bodies. This is comparable to antibody-based localizations and very different from transient IQCB1 overexpression, that results in a near ubiquitous distribution throughout the cytosol.
Ectopic overexpression of IQCB1 disperses its cellular localization and clouds its molecular interaction pattern in cultured mammalian cells. Utilizing endogenous loci tagging in cell models of retinal ciliopathies will provide much more accurate and powerful tools to study the specific molecular mechanisms of disease.
This is a 2020 ARVO Annual Meeting abstract.
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