Saliba, Richard Sebastian; (2002) Rhodopsin retinitis pigmentosa: An in vitro study of the cellular fate of wild type and mutant rhodopsin. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Mutations in the photopigment, rhodopsin, are the major cause of autosomal dominant retinitis pigmentosa. The majority of mutations in rhodopsin lead to misfolding of the protein. Through the detailed examination of P23H and K296E mutant opsin processing in COS-7 cells, I have shown that the mutant protein does not accumulate in the Golgi, as previously thought, but that it forms aggregates that have many of the characteristic features of an aggresome. The aggregates form close to the centrosome and lead to the dispersal of the Golgi apparatus. Furthermore, these aggregates are ubiquitinated, recruit cellular chaperones and disrupt the intermediate filament network. Mutant opsin expression can disrupt the processing of normal opsin, as co-transfection revealed that the wild-type protein is recruited to mutant opsin aggregates. In SH-SY5Y cells mutant opsin forms multiple aggregates, which are ubiquitinated, but aggresomes are rarely seen. The degradation of mutant opsin is dependent on the proteasome machinery. Unlike the situation with ?F508-CFTR, proteasome inhibition does not lead to a marked increase in aggresome formation in COS-7 cells but increases the levels of mutant opsin within the ER, suggesting that the proteasome is required for the efficient retro-translocation of the mutant protein. Inhibition of N-linked glycosylation with tunicamycin, leads to the selective retention of the mutant protein within the ER and increases the steady state level of mutant opsin. Glycosylation, however, has no influence on the biogenesis and targeting of wild- type opsin in cultured cells. This demonstrates that N-linked glycosylation is required for ER associated degradation of the mutant protein but is not essential for the quality control of opsin folding. The addition of 9-cis-retinal to the media increased the amount of P23H, but not K296E, that was soluble and reached the plasma membrane. The expression of mutant opsin in COS-7 cells appears to induce the upregulation of the ER resident chaperone BiP, which is known to be induced upon ER stress. Thus, it appears that the expression of mutant opsin induces an unfolded protein response. These data show that rhodopsin autosomal dominant retinitis pigmentosa is similar to many other neurodegenerative diseases in which the formation of intracellular protein aggregates is linked to disease pathogenesis and suggest a mechanism for disease dominance.

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