Molecular mechanisms of TIMP-3 turnover in Sorsby Fundus Dystrophy

Betts, Jacob Henry John (2024) Molecular mechanisms of TIMP-3 turnover in Sorsby Fundus Dystrophy. Doctoral thesis, University of East Anglia.

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Abstract

Sorsby fundus dystrophy (SFD) is an autosomal dominant macular dystrophy, caused by mutations in tissue inhibitor of metalloproteinases 3 (TIMP-3), leading to central vision loss. SFD is characterised by extracellular accumulation of mutant TIMP-3 proteins in the Bruch’s membrane (BrM), although molecular mechanism(s) facilitating this accumulation are unknown. In other cell types, extracellular levels of TIMP-3 are regulated post-translationally by the balance between its endocytosis via low-density lipoprotein receptor-related protein 1 (LRP1) and extracellular binding to heparan sulfate (HS).

To establish how SFD TIMP-3 proteins accumulate extracellularly, I investigated whether retinal pigment epithelial (RPE) cells endocytose wild-type (WT) and SFD variants (H181R, Y191C, S204C) of TIMP-3, and whether this occurs via LRP1. WT and SFD TIMP-3 were recombinantly expressed in HEK-293 cells and isolated by affinity chromatography. Endocytosis of these proteins from conditioned media of ARPE-19 and hTERT RPE-1 cells was assessed by immunoblotting and confocal microscopy. Endocytosis of Y191C and S204C was significantly slower than WT TIMP-3, whereas H181R had unimpaired endocytosis. Receptor-associated protein (RAP) blocked uptake of WT and SFD TIMP-3, however siRNA knockdown of LRP1 had no effect on WT TIMP-3 uptake. Endocytosis assays performed in LRP1-null mouse fibroblasts demonstrated that endocytosis of SFD TIMP-3 proteins is largely independent of LRP1. This suggests that RPE cells are distinct from other cells analysed to date, in that they endocytose WT and SFD TIMP-3 via an LRP other than LRP1.

Delayed endocytosis of Y191C and S204C TIMP-3 is likely a key molecular mechanism by which they accumulate in the BrM. However, the unimpaired endocytosis of H181R indicates heterogeneity in SFD molecular mechanisms. Further studies are needed to clarify binding of SFD TIMP-3 to HS, as this may be an alternative mechanism of accumulation. Targeting SFD TIMP-3 endocytosis or clearance may be a potential therapeutic approach to treat SFD.

Item Type:	Thesis (Doctoral)
Faculty \ School:	Faculty of Medicine and Health Sciences > Norwich Medical School
Depositing User:	Chris White
Date Deposited:	13 Aug 2024 07:38
Last Modified:	13 Aug 2024 07:38
URI:	https://ueaeprints.uea.ac.uk/id/eprint/96214
DOI:

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