Regulation, signaling, and dynamics of glucan phosphatases.

描述（由申请人提供）：本资助的目的是阐明葡聚糖磷酸酶laforin的调控、下游信号传导和结构动力学。哺乳动物细胞以糖原（一种水溶性碳水化合物）的形式储存易于调动的能量。编码E3泛素连接酶malin或双特异性磷酸酶laforin的基因的隐性突变破坏糖原代谢，导致致命的神经退行性癫痫，称为Lafora病（LD）。LD的一个标志是分支不良、过度磷酸化的不溶性碳水化合物积聚，称为Lafora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，泛素化并触发参与糖原代谢的多种蛋白质的降解。此外，我们还发现，劳弗素是一类独特的磷酸酶的创始成员，这些磷酸酶可以使磷酸葡聚糖去磷酸化。这些结果使我们提出了导致LD的分子机制：1)由于糖原代谢蛋白的不平衡，即蛋白质水平没有得到适当维持，导致LBs中malin结果的丢失；2)由于葡聚糖过度磷酸化抑制了葡聚糖分支，去甲素的缺失导致了LBs；3)劳力素也作为malin的靶向亚基，因此劳力素的缺失破坏了一些malin定向的泛素化事件。虽然我们在确定LD的分子机制方面取得了重大进展，但我们缺乏对这些酶如何被调节的理解。这个建议将阐明我们目前知识的不足。我们最近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，我们将定义触发去甲素磷酸化的体内条件，并描述功能后果。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)使用纯化蛋白在体外检测laforin的功能，3)利用LD小鼠模型验证我们的结果。在目标2中，我们将采用类似的策略来确定泛素化对去甲素功能的影响。此外，我们将定义在malin定向泛素化中的作用，因为我们最近发现，laforin作为malin的靶向蛋白。在Aim 3中，我们将确定葡聚糖磷酸酶结构组分的扰动如何导致LD。我们将利用氢-氘交换质谱法来定义去甲素的结构动力学，并利用x射线晶体学来确定葡聚糖磷酸酶的结构。这个建议是建立在我们过去的发现和使用互补的方法来推进我们对细胞代谢，神经变性和癫痫的插入事件的理解。这项工作的完成将更好地理解这些复杂的事件，并将产生治疗癫痫和神经变性的见解。

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.