Regulation, signaling, and dynamics of glucan phosphatases

 描述(由申请人提供)：该项目的目标是利用拉福拉病提供的细胞代谢的独特窗口来确定正常的糖原代谢和过程中断时的疾病含义。Lafora病(LD)是五大进行性肌阵挛癫痫之一，是一种致命性、隐性神经退行性疾病，在出生后第二个十年表现为癫痫事件。LD的一个特征是胞浆内聚集着被称为拉福拉小体的、过度磷酸化的、不溶于水的糖原样颗粒。这些包涵体遍及全身，但由于神经元对能量扰动的敏感性，疾病是由急性神经毒性引起的。LD是功能丧失的结果，或者是导致葡聚糖磷酸酶Laforin或E3泛素连接酶Malin编码基因功能异常的突变。我们确定拉福林为葡聚糖磷酸酶家族的创始成员，即使糖原或淀粉去磷酸化的磷酸酶。需要拉福林结构来确定可逆糖原磷酸化如何影响糖原代谢，并确定拉福林突变导致LD的原因。我们还证明了Malin是一种E3泛素连接酶，并报道了Malin泛素化参与糖原合成的蛋白。然而，Malin并不促进这些酶的降解，目前还不清楚Malin如何影响糖原代谢，以及Malin突变导致LD的原因。虽然Laforin或Malin基因突变会导致LD，但人们越来越认识到，多种机制导致LD，一系列突变会导致不同程度的疾病进展。因为我们的工作已经定义了Laforin和Malin的分子功能，所以我们可以唯一地确定Laforin和Malin中LD突变的临床生物化学。因此，我们建议：1.确定拉福林的结构机制。我们将利用X射线结晶学结合结构导向突变和功能分析来确定拉福林如何与糖原结合，磷酸化糖原如何去磷酸化，拉福林在糖原代谢中的作用，以及拉福林在LD中的作用。2.明确Malin在LD和糖原代谢中的作用。我们使用多种方法鉴定了马林底物，确定了泛素化的类型，并定义了一种底物泛素化的后果。我们将定义驱动Malin底物泛素化的信号事件，事件的动力学，功能后果，以及错误调控如何导致LD。我们将利用细胞培养和小鼠模型来确定Malin在糖原代谢和LD中的作用。3.将当前的见解转化为针对患者的诊断和治疗。LD既有错义突变，也有过早终止密码子。我们的初步分析显示，并不是所有的点突变都会取消活性。我们将利用我们的生化工具来确定Laforin和Malin LD突变的突变特定机制，并使用我们最近开发的生物检测来探索治疗方案。

 DESCRIPTION (provided by applicant): 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.