Kosmaoglou, M. (2009) Using molecular chaperones to manipulate rhodopsin retinitis pigmentosa. Doctoral thesis, UCL (University College London).
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The experiments described in this thesis were designed in order to test the hypothesis that molecular chaperones are involved in the biogenesis of rhodopsin and may be used in the treatment of rhodopsin retinitis pigmentosa. Rhodopsin is the prototypical G-protein coupled receptor found at high concentration in the outer segments of rod photoreceptor cells. Rhodopsin is made up of the rod opsin apoprotein and 11-cis-retinal, the photoactive ligand. Rhodopsin initiates the phototransduction cascade under dim light conditions and mutations in its primary sequence have been linked to the neurodegenerative blinding disease, retinitis pigmentosa. Mutations such as P23H, cause the misfolding of the protein, resulting in its retention in the endoplasmic reticulum of heterologous expression systems and the inner segment of photoreceptor cells. Whilst selecting suitable modifiers, the subcellular compartments occupied by rhodopsin during its biogenesis and the chaperones resident in these, were considered. Calnexin is a central component of the quality control machinery in the endoplasmic reticulum. As calnexin has been widely documented to assist in the maturation of nascent glycoproteins, mouse embryonic fibroblast cells were used, which expressed a truncated version of calnexin, unable to bind client glycoproteins. The expression of rod opsin was compared in cells expressing truncated calnexin and in their wild-type counterparts, assessing the contribution of calnexin in the subcellular localization and biochemical profile of rod opsin. Calnexin was found to be dispensable for the maturation and folding of rod opsin. EDEM1, the ER-degradation enhancing mannosidase α-like 1 protein, has been shown to accelerate the degradation of misfolded glycoproteins, extracting these from futile folding attempts in the calnexin cycle. EDEM1 was found to enhance the degradation of P23H rod opsin and importantly, promoted the cell surface expression of any remaining P23H molecules which escaped degradation. The localization of EDEM1 in murine retina was determined to be within a subset of the inner segment and rhodopsin was found to form a physiological immune complex in porcine retina. The binding protein, BiP, associates with nascent proteins as these are translated and translocated in the ER lumen. A toxin that efficiently cleaves BiP in two fragments was used in order to probe the effects of BiP deletion on the biogenesis of wild-type and mutant rod opsin. Wild-type rod opsin was found retained in the endoplasmic reticulum in the absence of functional BiP and was misfolded as ubiquitin was recruited to the endoplasmic reticulum surface from a previous diffuse localization. Therefore BiP appears to be critical for maintaining rod opsin in a folding competent state. A chaperone on the cytoplasmic face of the endoplasmic reticulum, namely HSJ1b, has previously been shown to result in the stalling of wild-type and mutant rod opsin folding. We have investigated the effects of coexpressing CHIP, the carboxy-terminus of Hsp70-interacting protein. CHIP is an E3 ligase, which has been shown to present misfolded proteins to the proteasome for degradation, via an association with the Hsp70 machinery and HSJ1b initiates the process by stimulating ATP hydrolysis by Hsp70. In the presence of HSJ1b, expression of CHIP resulted in the degradation of rod opsin by the proteasome. Hence chaperones in the endoplasmic reticulum lumen and the cytoplasm can be used to manipulate mutant P23H rod opsin and may be used in the treatment of rhodopsin RP.
|Title:||Using molecular chaperones to manipulate rhodopsin retinitis pigmentosa|
|Additional information:||Authorisation for digitisation not received|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Brain Sciences > Institute of Ophthalmology|
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