Year of Publication

2013

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Medicine

Department

Molecular and Cellular Biochemistry

First Advisor

Dr. Matthew Shawn Gentry

Abstract

Lafora disease (LD) is a rare yet invariably fatal form of epilepsy characterized by progressive degeneration of the central nervous and motor systems and accumulation of insoluble glucans within cells. LD results from mutation of either the phosphatase laforin, an enzyme that dephosphorylates cellular glycogen, or the E3 ubiquitin ligase malin, the binding partner of laforin. Currently, there are no therapeutic options for LD, or reported methods by which the specific activity of glucan phosphatases such as laforin can be easily measured. To facilitate our translational studies, we developed an assay with which the glucan phosphatase activity of laforin as well as emerging members of the glucan phosphatase family can be characterized. We then adapted this assay for the detection of endogenous laforin activity from human and mouse tissue. This laforin bioassay will prove useful in the detection of functional laforin in LD patient tissue following the application of therapies to LD patients. We subsequently developed an in vitro readthrough reporter system in order to assess the efficacy of aminoglycosides in the readthrough of laforin and malin nonsense mutations. We found that although several laforin and malin nonsense mutations exhibited significant drug-induced readthrough, the location of the epitope tag used to detect readthrough products dramatically affected our readthrough results. Cell lines established from LD patients with nonsense mutations are thus required to accurately assess the efficacy of aminoglycosides as a therapeutic option for LD. Using hydrogen-deuterium exchange mass spectrometry (DXMS), we then gained insight into the molecular etiology of several point mutations in laforin that cause LD. We identified a novel motif in the phosphatase domain of laforin that shares homology with glycosyl hydrolases (GH) and appears to play a role in the interaction of laforin with glucans. We studied the impact of the Y294N and P301L LD mutations within this GH motif on glucan binding. Surprisingly, these mutations did not reduce glucan binding as expected, rather enhancing the binding of laforin to glucans. These findings elucidate the mechanism by which laforin interacts with and acts upon glucan substrates, providing a target for the development of therapeutic compounds.

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