July 2018
Volume 59, Issue 9
Open Access
ARVO Annual Meeting Abstract  |   July 2018
Heat Shock Protein-27 is an Element of the BBS5 Interactome in Photoreceptors
Author Affiliations & Notes
  • Clay Clay Smith
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Susan Bolch
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Donald Dugger
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Erena Livingston
    Ophthalmology, University of Florida, Gainesville, Florida, United States
  • Footnotes
    Commercial Relationships   Clay Smith, None; Susan Bolch, None; Donald Dugger, None; Erena Livingston, None
  • Footnotes
    Support  NIH EY14864, Overstreet Retinal Research, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science July 2018, Vol.59, 3056. doi:
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    • Get Citation

      Clay Clay Smith, Susan Bolch, Donald Dugger, Erena Livingston; Heat Shock Protein-27 is an Element of the BBS5 Interactome in Photoreceptors
      . Invest. Ophthalmol. Vis. Sci. 2018;59(9):3056.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Previous studies of the protein complex in photoreceptors identified nineteen proteins that appear to associate with Bardet-Biedl Syndrome 5 (BBS5). In this study, we analyzed these components to determine if there was a direct interaction with BBS5.

Methods : Photoreceptor outer segment extracts were sedimented on a sucrose 20-30% gradient and protein profiles identified by immunoblots. Proteins that co-sedimented with BBS5 were subsequently analyzed by co-expression in HEK-293 cells, followed by co-immunoprecipitation with BBS5. Direct interaction with BBS5 was assessed by reconstitution assays, using purified, heterologously expressed protein. Immunohistochemistry of retinal sections was used to assess co-localization with BBS5.

Results : To validate the proteins that potentially interact with BBS5 in outer segments, we performed co-sedimentation assays on a 20-30% sucrose gradient. Of the nineteen proteins identified in our mass spectrometric study to co-precipitate with BBS5, fifteen co-sedimented with BBS5 on the sucrose gradient. Each of these fifteen proteins was cloned with an N-terminal FLAG tag, and co-expressed with untagged BBS5 in HEK-293 cells. Immunoprecipitation of the FLAG-tagged proteins pulled down BBS5 only with arrestin-1 and HSP27. To investigate if this was a direct interaction, purified BBS5, HSP27, and arrestin-1 were labeled with separate fluorophores and were reconstituted in an in vitro pull down assay. Pull down of HSP27 and arrestin-1 was directly linked to BBS5. However, HSP27 pull down was not linked to arrestin-1. In tissue sections, HSP27 immunoreactivity was distributed throughout the retinal layers, but also showed significant co-localization with BBS5 along photoreceptor axonemes.

Conclusions : Our findings indicate a direct interaction between HSP27 and BBS5, an association that likely occurs along the photoreceptor axoneme. We hypothesize that HSP27 performs a chaperone function in this complex of proteins, helping to maintain the stability of this protein complex.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

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