The cellular and molecular characterisation of the Muscular Dystrophy protein dysferlin

Abstract

Mutations in the dysferlin gene cause a group of inherited muscular dystrophies, collectively known as dysferlinopathies. Dysferlinopathies are disabling muscle diseases caused by the absence or reduction of dysferlin in skeletal muscle. Dysferlin is a member of the ferlin protein family, characterised by the presence of seven Ca2+-regulated lipid-binding C2 domains. With multiple C2 domains that can fold and unfold dynamically in response to Ca2+ or phospholipids, dysferlin is a key mediator in the Ca2+-dependent vesicle fusion of membrane repair. The tandem C2 domains separated by linkers suggests dysferlin is a flexible protein, however, little is known regarding the overall conformation of dysferlin or the exact mechanism by which mutations in dysferlin result in disease. Our group has recently shown, following membrane injury, Ca2+ influx activates calpains which cleave dysferlin releasing a C-terminal mini-dysferlinC72 fragment that we hypothesise plays a specialised role in the vesicle fusion of membrane repair. As part of my project I sought to refine the calpain cleavage site within dysferlin and probe the structural conformation of dysferlin in situ. My studies reveal that calpains act upstream of dysferlin in the cascade of membrane repair events, to specifically cleave dysferlin and release a specialised module for Ca2+-dependent membrane repair. I provide insight into the structural topology of dysferlin in situ and demonstrate how patient missense variants exert local changes on dysferlin conformation. These studies provide a significant step towards a better understanding of dysferlin tertiary arrangement within the cell, how this could influence its role in membrane repair, and how this is altered by the destabilisation that results from patient missense variants.