Diversity Supplement for Benjamin Turner: Regulation, signaling, and dynamics of glucan phosphatases

This project focuses on and expands Sub-Aim 1B of the funded R01. This Sub-Aim is entitled: Deconstruct laforin and define its role in glycogen metabolism and Lafora disease. The laforin structure opens exciting new avenues for defining LD mutations at the atomic level. These data will serve as a platform, allowing us to understand how specific patient mutations result in LD. This structure, coupled with a suite of assays we have developed (described below), will allow us to define a patient’s specific structural mechanism for LD laforin mutations. Ben will perform master and perform a number of experimental protocols to define LD patient mutations at the atomic level. There are 33 laforin point mutations scattered between laforin’s CBM and DSP domain. The CBM is responsible for glycogen binding, and the DSP possesses phosphatase activity. In addition to these two functions, laforin interacts with other proteins and some mutations disrupt protein-protein interactions. Thus, there are four classes of laforin mutations: 1) destabilized protein, 2) disrupted glycogen binding, 3) decreased/abolished phosphatase activity, and 4) disrupted protein-protein interactions. Since establishing laforin as the first glucan phosphatase we have modified existing assays and developed novel assays to monitor its activity. This suite of assays allows us to probe the ability of laforin to both bind and dephosphorylate glycogen; the assays include multiple glycogen binding assays, generic phosphatase assays using small molecules, a phosphatase assay using the physiological substrate glycogen, and a physiologically relevant substrate where we radio-label the C3- and C6-position and define laforin’s activity against the C3- and C6-phosphorylated positions.