Abstract
Purpose: :
Mutations in rhodopsin underlie many types of retinitis pigmentosa (RP). The P23H rhodopsin mutation leads to rhodopsin misfolding, retention within the endoplasmic reticulum (ER), and ultimately photoreceptor cell death. The Unfolded Protein Response (UPR) encompasses a set of intracellular signaling pathways that control the protein folding functions of the ER. Transcriptional targets of UPR signaling include chaperones, endoplasmic reticulum-associated protein degradation and ubiquitin-proteasome system components, and ER structural and transport proteins. Here, we investigated how artificial activation of individual UPR signaling pathways affect the folding and maturation of mutant rhodopsin.
Methods: :
To study the link between UPR and rhodopsin, we developed chemical-genetic tools that enabled artificial activation of individual UPR signaling pathways in cell culture. For the UPR pathway controlled by the Ire1 gene, we introduced an amino acid substitution of isoleucine to glycine at position 642 (IRE1[I642G]) that enabled regulation of its activity by the small ATP-analog, 1NM-PP1. For the UPR pathway controlled by Perk, we used a chimeric form of PERK fused to FK506-binding protein whose activity could be regulated by the small dimerizing molecule, AP20187. For the UPR pathway controlled by Atf6, we created a doxycycline-inducible fragment of ATF6 bearing the DNA and trans-activating domains. We transfected wild-type and mutant rhodopsin into cells bearing these chemical-genetic constructs, artificially activated UPR signaling pathways, and then measured the effects on rhodopsin biosynthesis.
Results: :
We found that UPR significantly affected P23H rhodopsin protein biogenesis: 1) Artificial IRE1 signaling selectively reduced misfolded P23H rhodopsin protein levels. 2) Artificial PERK signaling significantly reduced P23H rhodopsin protein levels although levels of other proteins also fell. 3) Artificial ATF6 activity preferentially reduced P23H rhodopsin levels.
Conclusions: :
Our findings identify three molecular strategies to reduce misfolded rhodopsin protein levels in cell culture based on manipulation of distinct UPR signaling pathways. Selective UPR activation may be useful in reducing P23H rhodopsin levels in photoreceptors and ameliorating RP.
Keywords: retina • retinal degenerations: cell biology • opsins