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James S. Friedman, Philip A. Ruzycki, Akhila G. Satish, Anand Swaroop; The adRP Gene KLHL7 And Its Potential Role In Ubiquitination And Photoreceptor Degeneration. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5400.
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To elucidate the biological role of KLHL7 and pathogenic mechanisms underlying retinal degeneration caused by mutations in KLHL7. We recently identified the KLHL7 gene as a cause of autosomal dominant retinitis pigmentosa. KLHL7 is a member of the BBK family of proteins, several of these act as mediators between E3 ubiquitin ligases and ubiquitinated substrates. Ubiquitination is critical for targeting substrate proteins to the proteasome, lysosome and autophagosome. We hypothesize that mutations in KLHL7 affect homeostasis by altering ubiquitination of photoreceptor-specific proteins.
We are performing GST pull-down, mass spectrometry experiments to identify candidate interacting proteins. GST-KLHL7 and GST were purified from bacterial cell lysate by Glutathione Sepharose beads. Protein production was verified by immunoblot. Lysed C57BL/6J retinas were pre-cleared with glutathione beads before overnight incubation with GST-KLHL7 and GST bound affinity medium. Beads were washed and bound proteins eluted with trypsinized KLHL7 alone. We are also using yeast two-hybrid analysis to determine KLHL7 interactors. KLHL7 N-terminal, C-terminal and full length cDNA constructs for yeast two-hybrid screens have been generated. We are additionally making human adult retinal and developing retinal yeast two-hybrid libraries for this purpose. This approach will allow for rigorous, thorough testing of each interactor protein, examining both the strength and types of interactions KLHL7 may participate in.
Full length GST-KLHL7 has been bacterially expressed and used in pull-down experiments. Eluted bands observed by silver staining that were identified as being unique to GST-KLHL7 as compared to GST alone have been examined by mass spectrometry. KLHL7 interacting proteins include Myosin-9, Ncam1 and DDX1. Additional validations are in progress.
Ubiquitination is critical to maintaining an appropriate turnover of proteins in every cell. Mutations in KLHL7 are hypothesized to perturb this process, leading to retinal degeneration. Our results suggest three candidate substrates for KLHL7 mediated ubiquitination.
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