Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 298, Issue 8, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.jbc.2022.102266
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Funding
- National Institutes of Health (NIH) [R01GM129261, R01EY025214, R01HL122010, R01GM080403, R01DA046138, P30EY011373]
- Graduate Training Program in Quantitative and Chemical Biology at Indiana University [T32 GM109825]
- Visual Science Research Center Core at Case Western Reserve University
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The study conducted deep mutational scanning to compare the plasma membrane expression of 123 known pathogenic rhodopsin variants, identifying 69 retinopathy variants with diminished expression and an increase in expression in the presence of 9-cis-retinal. The response to retinal varied considerably across the mutations spectrum, suggesting underlying differences in stability. Evaluation also showed that some variants compromised binding, but two of the previously uncharacterized variants retained residual function in vitro.
Over 100 mutations in the rhodopsin gene have been linked to a spectrum of retinopathies that include retinitis pigmentosa and congenital stationary night blindness. Though most of these variants exhibit a loss of function, the molecular defects caused by these underlying mutations vary considerably. In this work, we utilize deep mutational scanning to quantitatively compare the plasma membrane expression of 123 known pathogenic rhodopsin variants in the presence and absence of the stabilizing cofactor 9-cis-retinal. We identify 69 retinopathy variants, including 20 previously uncharacterized variants, that exhibit diminished plasma membrane expression in HEK293T cells. Of these apparent class II variants, 67 exhibit a measurable increase in expression in the presence of 9-cisretinal. However, the magnitude of the response to this molecule varies considerably across this spectrum of mutations. Evaluation of the observed shifts relative to thermodynamic estimates for the coupling between binding and folding suggests underlying differences in stability constrains the magnitude of their response to retinal. Nevertheless, estimates from computational modeling suggest that many of the least sensitive variants also directly compromise binding. Finally, we evaluate the functional properties of three previous uncharacterized, retinal-sensitive variants (Delta N73, S131P, and R135G) and show that two of these retain residual function in vitro. Together, our results provide a comprehensive experimental characterization of the proteostatic properties of retinopathy variants and their response to retinal.
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