RESUMO
Mutations in WHRN lead to Usher syndrome type 2d or to non-syndromic hearing impairment. The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2-associated proteins, usherin and ADGRV1. In photoreceptor cells, this protein complex constitutes fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism(s) of retinal degeneration due to compromised formation and function of the USH2-associated protein complex remains elusive. To unravel this pathogenic mechanism, we isolated and characterized 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 identified 19 novel candidate associated proteins. 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 indicates a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the newly identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients. SIGNIFICANCE: Since protein-protein interactions identified using targeted in vitro studies do not always recapitulate interactions that are functionally relevant in vivo, we established a transgenic zebrafish line that stably expresses a Strep/FLAG-tagged ortholog of human whirlin (SF-Whrna) in photoreceptor cells. Affinity purification of in vivo-assembled SF-Whrna-associated protein complexes from retinal lysates followed by mass spectrometry, identified 19 novel candidate interaction partners, many of which are enriched in the synaptic proteome. Two human orthologs of the identified candidate interaction partners, FRMPD4 and Kir2.3, were validated as direct interaction partners of human whirlin using a yeast two-hybrid assay. The strong connection of whirlin with postsynaptic density proteins was not identified in previous in vitro protein-protein interaction assays, presumably due to the absence of a biologically relevant context. Isolation and identification of in vivo-assembled whirlin-associated protein complexes from the tissue of interest is therefore a powerful methodology to obtain novel insight into tissue specific protein-protein interactions and has the potential to improve significantly our understanding of the function of whirlin and the molecular pathogenesis underlying Usher syndrome type 2.
Assuntos
Síndromes de Usher , Adulto , Animais , Humanos , Proteínas de Membrana/metabolismo , Proteoma/metabolismo , Retina/metabolismo , Síndromes de Usher/genética , Síndromes de Usher/metabolismo , Peixe-Zebra/metabolismoRESUMO
A combination treatment of shear, micro-bubbles, and high-frequency low-power ultrasound introduced via side-stream treatment of industrial water systems has shown excellent results in controlling bacteria and algae. Through the physical, high-stress environment created by ultrasonic waves, sessile and planktonic biological populations, some of which may undergo programmed cell death (PCD), can be controlled. Additionally, the instability and reduction of biofilm have been observed in systems treated by ultrasound and may be attributed to starvation-stress and lack of available cross-linking cations in the biofilm.