Defining the Role of Laforin in Glycogen Metabolism and Lafora Disease

项目总结/摘要 本提案的目标是利用拉福拉病（LD）提供的独特窗口来了解 laforin如何调节糖原代谢和定义LD患者的分子扰动 突变。Lafora病（LD）是五种主要的进行性肌阵挛癫痫之一，是一种致命的，遗传的， 在青春期出现的神经紊乱，总是导致神经退化和死亡。 LD患者显示过度磷酸化、异常分支的糖原样颗粒积聚 Lafora bodies（LB）LB存在于大多数组织中，但在LD中以神经症状为主 患者编码糖原磷酸酶laforin的基因突变导致大约70%的LD 例 LD主要以糖原代谢为中心。糖原是最重要的碳水化合物储存 在哺乳动物中，糖原是一种高分子，并且糖原的失调与许多疾病有关。近年来研究表明 糖原在脑功能中起动态作用，并且神经元对糖原扰动高度敏感。 虽然糖原已经研究了很多年，但我们对其调节的理解仍然存在差距。的 Gentry实验室将laforin确立为葡聚糖磷酸酶家族的创始成员（即， 从碳水化合物中释放磷酸盐），对LD的研究表明，缺乏laforin会触发 糖原转化为神经毒性碳水化合物，构成LB。作为唯一的葡聚糖 在人类中，laforin是通过磷酸化调节糖原的关键。 我们最近确定了拉福林的晶体结构。该提案将结合结构中的见解， 利用生物化学和生物物理学工具来定义laforin如何作为葡聚糖磷酸酶发挥作用， 糖原代谢此外，33个LD相关的错义突变分散在整个组织中。 结构和对laforin活性的不同影响。我们实验室的一个目标是确定所有这些因素的影响 突变。目标1A将解决二聚体界面的性质，这在该领域一直存在争议， 这一区域LD突变的分子效应。目标1B将定义laforin如何协同结合 糖原中的葡聚糖链以及长度和分支的影响。目标2将确立 laforin的结构域间区域（即磷酸酶和碳水化合物结合结构域之间）在维持 糖原的C3和C6特异性去磷酸化。许多LD突变落在该区域，并且该目的 将展示其功能效果。总之，本提案利用跨学科技术， 研究糖原稳态和LD的机制，研究将提供对大脑代谢的见解 并为针对患者的诊断和治疗铺平道路。

Project Summary/Abstract The goal of this proposal is to use the unique window offered by Lafora disease (LD) to understand how laforin regulates glycogen metabolism and define the molecular perturbation of LD patient mutations. Lafora disease (LD), one of the five major progressive myoclonus epilepsies, is a fatal, genetic, neurological disorder that manifests during adolescence and invariably leads to neurodegeneration and death. Patients with LD display accumulations of hyperphosphorylated, aberrantly branched, glycogen-like particles called Lafora bodies (LBs). LBs are found in most tissues but neurological symptoms predominate in LD patients. Mutations in the gene encoding the glycogen phosphatase laforin cause approximately 70% of LD cases. LD largely centers on glycogen metabolism. Glycogen is the most important carbohydrate storage molecule in mammals, and misregulation of glycogen is implicated in many diseases. Recent studies show that glycogen plays a dynamic role in brain function and that neurons are highly sensitive to glycogen perturbations. Although glycogen has been studied for many years, gaps in our understanding of its regulation remain. The Gentry lab established laforin as the founding member of the glucan phosphatase family (i.e. enzymes that release phosphate from carbohydrates), and studies on LD show that the absence of laforin triggers the transformation of glycogen into the neurotoxic carbohydrate that makes up LBs. As the only glucan phosphatase in humans, laforin is the lynchpin of glycogen regulation by phosphorylation. We recently determined the crystal structure of laforin. This proposal will couple insights from the structure with biochemical and biophysical tools to define how laforin functions as a glucan phosphatase and regulates glycogen metabolism. Further, the 33 LD-associated missense mutations are scattered throughout the structure and have different effects on laforin activity. A goal in our lab is to define the effect of all of these mutations. Aim 1A will address the nature of the dimer interface, which has been controversial in the field, and the molecular effect of LD mutations in this region. Aim 1B will define how laforin cooperatively binds the glucan chains in glycogen and the effects of length and branching. Aim 2 will establish the role of the interdomain region of laforin (i.e. between the phosphatase and carbohydrate-binding domains) in maintaining C3- and C6-specific dephosphorylation of glycogen. A number of LD mutations fall in this region and this aim will demonstrate their functional effects. In summary, this proposal utilizes cross-disciplinary techniques to study glycogen homeostasis and the mechanisms of LD, studies that will provide insights into brain metabolism and pave the way for patient-specific diagnoses and treatments.