Biology and enzymatic cleavage of ferlins: from membrane repair to cancer

Abstract

Enzymatic cleavage can modify the function of target proteins. In this thesis I investigate the enzymatic cleavage of myoferlin and dysferlin, two members of the ferlin family. In response to membrane injury, dysferlin is cleaved enzymatically by calpains-1 and -2, releasing a C-terminal mini-dysferlinC72 and an N-terminal counter fragment. My thesis provides supporting evidence that mini-dysferlinC72 functions as a specialised module in the emergency response of membrane repair. I investigate the calpain-dysferlin membrane repair axis in different types of membrane injury, and the effect of calpain knock-out in skeletal muscle in a murine model. Using CRISPR gene-edited single calpain-1 or calpain-2 knock-out cells, I reveal that calpains-1 and -2 can compensate for each other, leading to almost normal membrane repair outcomes; but when both ubiquitous calpains are missing, cells lose almost all of their Ca2+-dependent membrane repair capacity. The second part of my thesis aims to compare the cleavage of myoferlin to the calpain cleavage of dysferlin. The results of this study reveal that the cleavage of myoferlin is differently regulated to the calpain-cleavage of dysferlin. I show that the cleavage of myoferlin, unlike dysferlin cleavage, is independent of calpains-1 and -2, and is not a direct response of the Ca2+ flux of membrane injury. Mini-myoferlin can be detected at rest in different cell types, including breast cancer cell lines, as well as in human triple negative breast cancer tumour specimens. I investigate potential functional impact of enzymatic cleavage to release mini-myoferlin, and discover a link between myoferlin cleavage and cancer linked to upregulation of the MAPK/ERK signaling pathway.