Regulation, signaling, and dynamics of glucan phosphatases.

描述(由申请人提供)：这项资助的目的是阐明葡聚糖磷酸酶Laforin的调节、下游信号和结构动力学。哺乳动物细胞以糖原的形式储存容易动员的能量，糖原是一种水溶性碳水化合物。编码E3泛素连接酶Malin或双重特异性磷酸酶Laforin的基因的隐性突变会扰乱糖原代谢，并导致一种称为Lafora病(LD)的致命性神经退行性癫痫。LD的一个特征是分支不良、过度磷酸化的不溶性碳水化合物积累，称为拉福拉小体(Lbs)，被认为是LD的病原体。我们确定Malin是一种单亚基E3泛素连接酶，它泛素化并触发参与糖原代谢的多种蛋白质的降解。此外，我们发现拉福林是一类独特的磷酸酶的创始成员，该酶能使磷酸葡聚糖去磷酸化。这些结果使我们能够提出导致LD的分子机制：1)由于糖原代谢蛋白质的失衡，导致LBS中Malin的丢失，即蛋白质水平没有得到适当的维持；2)由于葡聚糖过度磷酸化，抑制葡聚糖分支，导致LBS中Laforin的丢失；3)Laforin也作为Malin的靶向亚基，因此Laforin的丢失扰乱了一些Malin导向的泛素化事件。虽然我们在确定LD的分子机制方面取得了重大进展，但我们对这些酶是如何调控的缺乏了解。这项提议将阐明我们目前知识中的不足之处。我们最近在Laforin上发现了新的磷酸化和泛素化事件。磷酸化和泛素化是翻译后修饰，直接改变蛋白质浓度、酶活性、蛋白质定位、蛋白质-蛋白质相互作用和结构动力学。在目标1中，我们将定义体内触发laforin磷酸化的条件，并表征其功能后果。我们将利用1)组织培养细胞中的过表达和内源性蛋白水平来监测Laforin的定位和修饰状态，2)使用纯化的蛋白在体外检测Laforin的功能，以及3)利用LD小鼠模型来验证我们的结果。在目标2中，我们将使用类似的策略来确定泛素化对Laforin功能的影响。此外，我们还将确定Laforin在Malin引导的泛素化中的作用，因为我们最近发现Laforin作为Malin的靶向蛋白。在目标3中，我们将确定葡聚糖磷酸酶结构成分的扰动是如何促进LD的。我们将利用氢-氚交换质谱学来确定Laforin的结构动力学，并用X射线结晶学来确定葡聚糖磷酸酶的结构。这一建议建立在我们过去的发现基础上，并使用互补的方法来促进我们对细胞代谢、神经退行性变和癫痫等中间事件的理解。这项工作的完成将使人们更好地了解这些复杂的事件，并将产生对癫痫和神经变性的治疗见解。

DESCRIPTION (provided by applicant): The objective of this grant is to elucidate the regulation, downstream signaling, and structural dynamics of the glucan phosphatase laforin. Mammalian cells store readily mobilized energy in the form of glycogen, a water-soluble carbohydrate. Recessive mutations in genes encoding the E3 ubiquitin ligase malin or the dual specificity phosphatase laforin disrupt glycogen metabolism and result in a fatal, neurodegenerative epilepsy called Lafora disease (LD). A hallmark of LD is poorly branched, hyperphosphorylated insoluble carbohydrate accumulations called Lafora bodies (LBs), thought to be the causative agent of LD. We established that malin is a single-subunit E3 ubiquitin ligase that ubiquitinates and triggers the degradation of multiple proteins involved in glycogen metabolism. In addition, we discovered that laforin is the founding member of a unique class of phosphatases that dephosphorylate phospho-glucans. These results allowed us to propose molecular mechanisms that cause LD: 1) loss of malin results in LBs due to an imbalance in glycogen metabolism proteins, i.e. protein levels are not properly maintained; 2) loss of laforin results in LBs due to glucan hyperphosphorylation that inhibits glucan branching; 3) laforin also acts as a targeting subunit for malin, so that loss of laforin disrupts some malin-directed ubiquitination events. While we have made significant strides in determining the molecular mechanisms of LD, we lack an understanding of how these enzymes are regulated. This proposal will elucidate the deficiencies in our current knowledge. We recently identified novel phosphorylation and ubiquitination events on laforin. Phosphorylation and ubiquitination are post-translational modifications that direct changes in protein concentration, enzymatic activity, protein localization, protein-protein interactions, and structural dynamics. In Aim 1 we will define the in vivo conditions triggering laforin phosphorylation and characterize the functional consequences. We will utilize 1) overexpression and endogenous protein levels in tissue culture cells to monitor the status of laforin localization and modification, 2) assay laforin function in vitro using purified proteins, and 3) utilize a LD mouse model to verify our results. In Aim 2, we will utilize a similar strategy to determine the affects of ubiquitination on laforin function. In addition, we will define the role of laforin in malin directed ubiquitination, as we have recently discovered that laforin acts as a targeting protein for malin. In Aim 3, we will determine how perturbations of the structural components of glucan phosphatases contribute to LD. We will utilize Hydrogen- Deuterium exchange mass spectrometry to define the structural dynamics of laforin and x-ray crystallography to determine the structure of a glucan phosphatase. This proposal is built on our past discoveries and uses complementary approaches to advance our understanding of the intercalated events of cell metabolism, neurodegeneration, and epilepsy. Completion of this work will yield a better understanding of these complex events and will produce therapeutic insights for epilepsy and neurodegeneration.