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bioRxiv · 10.1101/2024.10.23.619933

Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling

Solarat, C.·Barra-Carneiro, J.·Vila-Almuina, S.·Barbeito, P.·Holm, M. S.·Gammoh, N.·Christensen, S. T.·Valverde, D.
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Abstract

Background: Ciliopathies are genetic disorders caused by defects in the structure or function of the cilia and their related structures. Bardet-Biedl syndrome (BBS) is a complex ciliopathy with varied symptoms, probably due to altered membrane receptor signalling pathways. Methods: We investigated the role of BBS1 and BBS4 gene deficiencies in retinal epithelial cells using surface membrane proteomics, co-immunoprecipitation, AlphaFold structural modelling, endocytic trafficking colocalization assays, autophagy flux analysis, and epithelial-to-mesenchymal transition (EMT) functional assays including migration and wound healing. Results: Deficiencies in these BBSome components led to shorter cilia and disrupted receptor endocytosis, with accumulation of TGF-{beta}-pathway and EMT-associated proteins on the plasma membrane of BBS1 KO cells. Co-immunoprecipitation and AlphaFold structural modelling indicated that neither BBS1 nor BBS4 forms stable physical complexes with core endocytic components (EEA1, RAB11, RAB7, LAMP2), suggesting an indirect mechanism. BBS1 KO cells showed sustained TGFBR1 recycling, low basal autophagic flux that increases upon TGF-{beta} stimulation, and the most pronounced mesenchymal phenotype. BBS4 KO cells showed reduced receptor recycling, elevated basal autophagic flux that declines upon stimulation, and the strongest canonical TGF-{beta}/SMAD activation with reduced non-canonical ERK1/2 signalling. Conclusions: Increased EMT markers and enhanced migration in BBS1 KO cells highlight the role of BBSome-dependent receptor trafficking as a potential therapeutic target for retinal degeneration in BBS. The divergent autophagic and signalling phenotypes between BBS1 and BBS4 KO reveal subunit-specific contributions to TGF-{beta} pathway dysregulation.

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Solarat, C., Barra-Carneiro, J., Vila-Almuina, S., Barbeito, P., Holm, M. S., Gammoh, N., Christensen, S. T., Valverde, D.. 2024-10-24. Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling. https://doi.org/10.1101/2024.10.23.619933

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