Supplement: Regulation, Signaling, and Dynamics of Glucan Phosphatases

The goal of this project is to utilize the unique window into cellular metabolism that Lafora disease offers to define both normal glycogen metabolism and disease implications when the process is disrupted. Lafora disease (LD), one of five major progressive myoclonic epilepsies, is a fatal, recessive neurodegenerative disorder that presents as an epileptic event in the 2nd decade of life. A hallmark of LD is the accumulation of cytoplasmic, hyperphosphorylated, water-insoluble glycogen-like particles called Lafora bodies. These inclusions occur throughout the body, but disease results from acute neurotoxicity due to the sensitivity of neurons to energy perturbations. LD is the result of loss of function mutations or mutations that cause aberrant function in either of the genes encoding the glucan phosphatase laforin or E3 ubiquitin ligase malin. We established laforin as the founding member of the glucan phosphatase family, i.e. phosphatases that dephosphorylate glycogen or starch. A laforin structure is needed to determine how reversible glycogen phosphorylation impacts glycogen metabolism and define why laforin mutations result in LD. We also demonstrated that malin is an E3 ubiquitin ligase and reported that malin ubiquitinates proteins involved in glycogen synthesis. However, malin does not promote degradation of these enzymes and it has remained unknown as to how malin impacts glycogen metabolism and why mutations in malin result in LD. While mutations in the laforin or malin gene result in LD, it is increasingly recognized that multiple mechanisms lead to LD and that a spectrum of mutations yields different degrees of disease progression. Because our work has defined the molecular function of laforin and malin, we are uniquely poised to define the clinical biochemistry of LD mutations in both laforin and malin. Therefore, we propose to: 1. Determine the structural mechanism of laforin. We will utilize X-ray crystallography combined with structure-guided mutagenesis and functional assays to determine how laforin binds to glycogen, how phosphorylated glycogen is dephosphorylated, laforin's role in glycogen metabolism, and laforin's role in LD. 2. Define the role of malin in LD and glycogen metabolism. Using multiple methods we have identified malin substrates, and established the type of ubiquitination as well as defined the consequences of ubiquitination for one substrate. We will define the signaling events that drive ubiquitination of malin substrates, the dynamics of the events, the functional consequences, and how misregulation leads to LD. We will utilize cell culture and mouse models to determine the role of malin in glycogen metabolism and LD. 3. Translate current insights into patient-specific diagnosis and treatment. LD results from both missense mutations as well as Premature Termination Codons. Our initial analysis revealed that not all point mutations abolish activity. We will utilize our biochemical tools to define mutation specific mechanisms for laforin and malin LD mutations and explore therapeutic options using our recently developed bioassay.