Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina - GST
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ABSTRACT: Mutations in WHRN lead to Usher syndrome type 2d (USH2d) or to non-syndromic hearing impairment (DFNB31). The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2 (USH2)-associated proteins, usherin and ADGRV1. Together with PDZD7, these proteins form the transiently present ankle link complex necessary for maturation of the inner ear hair bundle. In photoreceptors, usherin and ADGRV1 are also scaffolded by whirlin and constitute similar fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism of retinal degeneration as a consequence of compromised USH2-associated protein complex formation and function remains elusive. To better understand the function of the USH2-associated proteins in the photoreceptors, we aimed to isolate and characterize whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry analysis identified 19 novel candidate interaction partners. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which suggest a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients.
INSTRUMENT(S): Orbitrap Fusion
ORGANISM(S): Danio Rerio (zebrafish) (brachydanio Rerio)
TISSUE(S): Layer Of Retina, Retina
DISEASE(S): Disease Free
SUBMITTER: Karsten Boldt
LAB HEAD: Dr. Karsten Boldt
PROVIDER: PXD034247 | Pride | 2023-12-28
REPOSITORIES: Pride
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