Intracellular functions and interactions of age-related macular degeneration-associated variant B cystatin C.

Myerscough, Christopher
Intracellular functions and interactions of age-related macular degeneration-associated variant B cystatin C. PhD thesis, University of Liverpool.

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Age related macular degeneration (AMD) is the leading cause of blindness among the elderly population. It occurs in two forms, a nonexudative "dry" form which can lead to geographic atrophy of the retinal pigment epithelial (RPE) cells in the region of the macula and an exudative "wet" form which involves neovascularisation from the choroid, through Bruch's membrane and the RPE layer and into the photoreceptor layer causing significant damage. Cystatin C is a cysteine protease inhibitor known to inhibit cathepsin activity. It is a highly abundant transcript in the RPE and is known to be processed through the secretory pathway . A variant form of this protein with an amino acid substitution in its signal sequence results in the protein being retained intracellularly and is found localised with the mitochondria. This variant form has been associated with increased risk of developing exudative AMD. In this study a number of key cellular processes were examined to elucidate the effect the variant B protein has on the RPE. These processes were respiration, apoptosis, autophagy and oxidative stress; all of which have been implicated in AMD pathology or ageing. In addition the protein-protein interactions of variant B cystatin C were assessed through mass spectrometry analysis of pulled-down cystatin C protein from transfected cell lysates. No effect on respiration, apoptosis or autophagy was identified. However a statistically significant difference in oxidative stress was identified as a result of overexpression of either wild type cystatin C or variant B cystatin C. Mass spectrometry analysis resulted in two highly promising proteins that were found interacting with variant B cystatin C at a statistically significant level, prohibitin and voltage-dependent anion-selective channel protein 1 (VDAC1). Although oxidative stress was found for both proteins given the nature of the expression (driven by a CMV promoter) it seems likely that the oxidative stress response is due to a high level of intracellular cystatin C. This suggests that the retention of variant B protein within the cell would lead to increased oxidative stress levels. It can be speculated that this response could be a contributory factor to development of AMD. VDAC1 and prohibitin may offer an explanation of what happens to the variant B protein within RPE cells. VDAC1 is the major pore-forming protein in the mitochondrial membrane, disruption of its functioning by the binding of variant B protein might be expected to have detrimental effects. Prohibitin is an even more promising target as it has been associated with oxidative stress. In addition it is known to translocate between the nucleus and the mitochondria, offering the tantalising possibility of a complete explanation for the mitochondrial mislocalisation.

Item Type: Thesis (PhD)
Additional Information: Date: 2015-06-23 (completed)
Subjects: ?? Q1 ??
Depositing User: Symplectic Admin
Date Deposited: 30 Jul 2015 07:25
Last Modified: 17 Dec 2022 01:18
DOI: 10.17638/02014500