The degradation of mutant RhodopsinIle256del follows the VCP‐dependent endoplasmic‐reticulum‐associated protein degradation pathway

Abstract

AbstractPurposeIn Europe, the Rholle256del mutation (deletion of isoleucine at position 255 or 256 in Rho gene) accounts for over 17% of familial ADRP cases (C. F. Inglehearn, 1991). From a clinical perspective, the severity of this mutation can be compared to RhoP23H. In previous studies, we found that the degeneration of misfolded RHOP23H follows the ERAD pathway and the clearance of RHOP23H requires the ERAD effector valosin‐containing protein (VCP). This study aims to characterize the subcellular structural and functional changes in Rholle256del transfected mammalian cells, evaluate VCP inhibition's effects, and understand the pathological similarity between Rholle256del and RhoP23H transfected cells.MethodsHEK293 and COS‐7 cells were transfected with plasmids encoding RhoWT, Rholle256del, or RhoP23H, tagged with GFP. Immunostaining was performed to reveal the localization of transfected RHO‐GFP and the co‐localization between RHO‐GFP with ER, endogenous VCP, ubiquitin, and proteasome subunit (PSMB5). Formation and degree of Rho protein aggregates were analyzed in transfected cells before and after proteasome‐ and VCP‐inhibition.ResultsWe found that RHOWT mainly localizes at the plasma membrane, while mutant RHOlle256del and RHOP23H accumulates in the cytosol forming cytoplasmic aggregates. Co‐staining results suggest that both RHOlle256del and RHOP23H aggregates are retained within the ER, partially co‐localizing with VCP. RHOlle256del and RHOP23H aggregates were ubiquitinated and partially transported to the proteasome system for degradation. VCP inhibition led to enhanced accumulation of high molecular weight (HMW) RHO‐containing oligomers and aggregates.ConclusionsOur results indicate similar clearance characteristics between Rholle256del and RhoP23H in transfected cells, including the excessive formation of misfolded RHO aggregates, retention in the ER, and, at least partially, interaction with VCP. Moreover, VCP inhibition affects the abundance of RHO aggregates. This suggests that the clearance of RHOlle256del also follows the VCP/ERAD/proteasome axis, raising a possibility that modulation of VCP could be considered as a potential therapy for Rholle256del‐related RP as well.